EP2445538A2 - Siarn ciblé contre un aptamère pour inhiber une dégradation à médiation par un non-sens - Google Patents

Siarn ciblé contre un aptamère pour inhiber une dégradation à médiation par un non-sens

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Publication number
EP2445538A2
EP2445538A2 EP10797598A EP10797598A EP2445538A2 EP 2445538 A2 EP2445538 A2 EP 2445538A2 EP 10797598 A EP10797598 A EP 10797598A EP 10797598 A EP10797598 A EP 10797598A EP 2445538 A2 EP2445538 A2 EP 2445538A2
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Prior art keywords
cell
aptamer
molecule
target
oligonucleotide
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German (de)
English (en)
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Eli Gilboa
Fernando Pastor
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University of Miami
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University of Miami
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0091—Purification or manufacturing processes for gene therapy compositions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering nucleic acids [NA]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/16—Aptamers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/35—Nature of the modification
    • C12N2310/351—Conjugate
    • C12N2310/3513—Protein; Peptide
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/35—Nature of the modification
    • C12N2310/351—Conjugate
    • C12N2310/3519—Fusion with another nucleic acid

Definitions

  • Embodiments of the invention provide compositions and methods for highly selective targeting of heterologous nucleic acid sequences.
  • the heterologous nucleic acid sequences comprise oligonucleotides, for example, short interfering RNA's (siRNA's) which are targeted to desired cells in vivo and which bind in a sequence dependent manner Io their targets.
  • siRNA's short interfering RNA's
  • Embodiments of the Invention comprise the generation of multi-domain molecules comprising a target specific domain and at least one domain, which modulates expression and function of molecules associated with nonsense mediated decay pathways.
  • the multi-domain molecules comprise aptam ⁇ r- oligonucleotides for specifically targeting an oligonucleotide, e.g. interference RNA (RNAi) to a desired cell in vivo.
  • RNAi interference RNA
  • the aptamers are generated against specific products expressed by a target cell and the oligonucleotides arc specific for the nonsense mediated decay pathway and associated molecules. Inhibition of the nonsense mediated decay pathway allows for the up- regulation of existing antigens and/or the induction of new antigens not previously expressed on the target cells and/or novel antigens which results in the induction or enhancement of antigen city of a the target cell ultimately leading to its destruction by the immune system.
  • Methods of treating a patient comprise administration of a therapeutically effective amount of chimeric molecules, such as for example, aplamer-oligonucleotide molecules.
  • FIG. 1 is a schematic representation showing the mechanism by which the NMD process prevents the accumulation of premature termination codon (PTC) containing mllNAs in ⁇ ukaryotic cells. Removal of introns from the pr ⁇ -mRNA leaves behind an exon-junction complex (EJC) demarcating the splice junctions (Panel A). An NMD complex consisting of several factors including Upfl, Upf2 and Up3 is then assembled on each EJC as shown in Panel B. SMG 1, which phosphorylales Upfl , and Upfl are the two key rate limiting factors in the formation of the complex.
  • PTC premature termination codon
  • EJC exon-junction complex
  • the EJCzNMD complex When the mRN A undergoes the first round of translation, called the "pioneer translation", the EJCzNMD complex is removed, presumably as a result of the translational machinery moving thru the region, thereby rendering the mRNA stable and competent for additional rounds of translation. If a PTC is present in an exon (other than the last ⁇ xon), for example as a result of an infrarae nonsense mutation, the EJC/NMD complexes downstream to the PTC arc not removed from the mRN A. The attached NMD complex then triggers the degradation of the mRNA.
  • FIG. 2 is a schematic representation showing the principle of targeted inhibition of NMD in tumor cells using aptamcr targeted siRNAs.
  • An aptamcr which was selected to bind to a tumor-specific cell surface product, such as PSMA expressed on prostate cancer cells, is conjugated to an siRNA corresponding to an ISIMD factor such as SMG- i or UpC (see below).
  • the systcmically administered aptamcr-oligonucleotidcs homes Io and delivers the siRNA to the tumor cells expressing the cognate receptor.
  • FIG. 3 shows that a PSMA aptamer -- SMG-I siRNA chimera downregulates SMG- 1 RNA expression in PSMA-exprcssing tumor cells in a PSM A-dependent manner.
  • Inhibition of SMG-I RNA by SMG-I siRNA and PSMA apamer-SMG-1 siRNA chimera shows that conjugation of the siRNA to the aptamer did not compromise its functionality.
  • Inhibition of SMG-I RNA by the PSMA apamer-SMG-1 siRNA chimera in the absence of lipofectamine in PSMA-CT26 cells but not CT26 cells shows that the SMG-I siRNA was targeted to CT26 cells in a PSM A-dependent manner.
  • F igures 4 A-4C show the expression of UpJ2 or Smgl shRN A in CT26 tumor cells leads to immune-mediated inhibition of tumor growth
  • Figure 4 A Intratumoral accumulation of OVA-specific OT-I T cells in response to NMD inhibition.
  • B16/F 10 tumour cells transduced with shRNA-cncoding lenti viral vectors (described in Figure 8A) were stably transfcctcd with an NMD reporter plasrnid (described in Figure 8B) containing the class I-restricted epitope of chicken ovalbumin (OVA).
  • mice were implanted subcutaneously with parental tumor cells (wild-type (WT) B 16) or with the lentivirus-transduced tumor cells, and either received or did not receive doxycycline in their drinking water.
  • parental tumor cells wild-type (WT) B 16
  • lentivirus-transduced tumor cells lentivirus-transduced tumor cells
  • FIG. 4B Balb/ ' c mice were implanted subcutaneously with CT26 tumor cells stably transduced with the shRNA inducible lentivirai vector encoding SmgL Up/2 and control shRNA (ten mice per group). Each group was divided into two subgroups receiving (filled circles) or not receiving (open circles) doxycycline in the drinking water, n zzz 2.
  • Figure 4C Same as Figure 4B except that tumor ceils were injected into immunc-dcficicnt nude mice.
  • Figures 5A-5C arc graphs showing the inhibition of tumor growth in mice treated with PSMA aptamcr targeted Up/2 and Smgl siRNAs.
  • Figure 5B shows that provides a tumor growth in mice treated with PSMA aptamcr targeted Up/2 and Smgl siRNAs.
  • Figure 5B Balb/c mice were implanted subcutaneously with PSMA-CT26 tumor cells and 3 days later injected via the tail vein with PBS (filled circles) or with PSMA aptamcr-siRNA conjugates (open circles, control siRNA; open squares, Up/2 siRNA; rilled squares, Sm
  • mice C57BL/6 mice were implanted with PSM A-B 16/Fl 0 tumor cells by tail vein injection, and 5 days later were injected with PSMA aptamcr-siRNA conjugates (ten mice per group).
  • PSMA-CT26 tumor-bearing mice five mice per group were treated with various combinations of PSMA aptamcr conjugated to Smgl or control siRNA and an agonistic or costimulation- deficient 4- IBB aptamer dinier (raut4-lBB) and monitored for tumor growth. /? ::: 1.
  • Figures 6A-6C PSMA aplamcr- ⁇ Vng/ siRNA rejection of PSMA-exprcssing, but not parental, CT26 tumor cells.
  • Figure 6A Mice were co-implanted subcutancousiy with PSMA- expressing (left flank) and parental (right (lank) CT26 tumor cells arid injected with PSMA aptamer --Smgl siRNA via the tail vein.
  • Figure tC Three days after tumor inoculation, mice were injected with aptamer-siRNA conjugate (eight mice per group) as described in Figure 5A and tumor growth was monitored. Open circles, parental CT26; filled circles, PSMA-CT26. n ' - ' - ' - 2.
  • FIGS. 8A-8B RNA downregulation and NMD inhibition in CT26 tumor cell stably expressing Upf-2 or SMG-I shRNA.
  • Colon carcinoma-derived CT26 tumor cells were stably transduced with the PTIG-UMetO ientiviral vector encoding Upf-2 and SMG-I shRNA.
  • PTIG- U ⁇ tetOshRlMA contains a U6-promotcr driven shRNA cassette which is under tet regulation (Diagram, Figure 8A), as well as a bicislronic CMV promoter-driven cassette encoding the tet repressor and EGFP) (not shown in the diagram).
  • Stably transduced cells were isolated by sorting for EGFP ' cells and grown in the presence or absence of doxycycline.
  • Figure 8A RNA downregulation and NMD inhibition in CT26 tumor cell stably expressing Upf-2 or SMG-I shRNA.
  • Colon carcinoma-derived CT26 tumor cells were stab
  • Upf-2 or SMG-I RNA is reduced in CT2e> tumor cells expressing the
  • shRNA-mcdiated Upf-2 or SMG-I downrcgulation leads to NMD inhibition.
  • NMD activity in the shRNA- expressing CT26 cells was determined by transiently transfccting the Upf-2 and SMG- 1 shRNA expressing CT26 cells with an NMD reporter plasmid encoding a ⁇ -globin gene which contains a premature termination codon (PTC?) in its second exon ( see diagram) or with a similar plasmid encoding the wild type (wt) ⁇ -globin gene.
  • Cells were grown in the presence or absence of doxycycline and the relative amounts of ⁇ -globin transcripts were determined by semiquantitative RT-PCR.
  • FIG. 9 Delaying SMG-I shRNA expression in tumor bearing mice diminishes tumor inhibition.
  • Balb/c mice were implanted subcutan ⁇ ously with the shRNA inducible lentiviral vector encoding SMG-I shRNA as described in Figure 4B, As indicated, doxycycline was provided in the drinking water at the day of tumor implantation (day 0) or delayed for 2, 4 or 6 days (5 mice per group).
  • FIGS 10A- 1OC Induction of T cell responses against NMD-controlled products. Mice were immunized with CT26 tumor cells transduced with the doxycycline-inducible SMG-I (SMG-I) or control (Control) shRNA (Figure 8A) in the presence doxycycline in the drinking water, and 5 or 30 days later splenocytes were isolated and enriched for cither total CD3 L T cells, CDS " T cells, or CD4 ⁇ r T cells.
  • Figure 1 OA Induction of immune responses against tumor cells in which NMD was inhibited.
  • Tumor antigens in the form of mRNA were isolated from SMG-I (SMG-I) or control (Control) shRNA transduced CT26 tumor cells cultured in the presence of doxycycline, and transf ⁇ cted into syngeneic dendritic ceils (DC?). T cells isolated 5 days after tumor implantation (respondcrs) were incubated with the mRNA transfecled DC (stimulators), and proliferation was determined 4 days later by measuring ' ⁇ -thymidine incorporation.
  • mRNA transfected DC as stimulators provides a useful way to compare the antigenicity of diverse cell populations and tissues that often exhibit significant variations in their background immunog ⁇ nicity, e.g., stimulation of T cell proliferation measured by 3 Hthymidin ⁇
  • Figure 1 OC Epitope spread - induction of immune responses against the parental tumor. Experimental conditions as described in Figure 1OA using total COV T cells as responders, except that T cells were isolated 5 day as well as 30 days post tumor implantation,
  • FIG 11 is a schematic representation showing the primary sequence and computer generated secondary structure of an embodiment of a PSMA aptamer-siRNA conjugates.
  • PSMA aplamer-siRNA fusions were generated corresponding to the SWAP configuration except thai two thymidine nucleotides were added to the 3' of the passenger strand to prevent dicer binding.
  • RNAstruclre 4.1 program was used for secondary structure analysis, PSMA aptamer - black; siR NA guide strand - blue; siRNA passenger strand - red.
  • Figure 12 Binding and uptake of PSMA aptamer-SMG-1 siRNA by PSMA- expressing CT26 tumor cells.
  • Parental and PSM A-expressing CT26 tumor cells were incubated with anti-PSMA antibody (green) or Cy 3 -conjugated PSMA aptamer-SMG-1 siRNA (pink) and analyzed by confocal microscopy (6OX magnification). Nuclei were stained with DAPI (blue).
  • FIG. 13 PSMA -dependent inhibition of SMG-I or U ⁇ f-2 RNA in PSMA-CT26 tumor cells incubated with PSMA aptamer-siRNA conjugates.
  • PSMA-CT26 or parental CT26 cells were incubated with SMG-I siRNA, control siRNA.
  • PSMA aptamer-SMG-1 siRNA or PSMA aptarner-eontrol siRNA left panel
  • PSMA aptaracr-Upf-2 siRNA arid PSMA- control siRNA right panel
  • SMG-I and Upf-2 RNA content was determined 48 hours later using semiquantitative RT-PCR.
  • the SMG-I siRNA retains its function when conjugated to the PSMA aplamer since free and conjugated siRNA transfccted in the presence of lipofectamine were comparably effective in downrcgulating SMG-I RNA (left panel), Inhibition by the aptarner conjugated siRNAs is PSMA dependent since in the absence of transfcction agent, incubation of PSMA-cxprcssing, but not parental, CT26 tumor cells with either PSMA aptamer-SMG-1 or Upf-2 siRNA conjugates led to downrcgulation of its target RNA.
  • Figures 14A-14B Inhibition of tumor growth and survival of CT26 tumor bearing mice injected with PSMA-SMG-I siRNA conjugates. Balb/c mice were implanted
  • PSMA aptanier-control siRNA conjugate Con
  • PSMA aptanier-control siRNA conjugate Con
  • PSMA aptamer-SMG-1 siRNA conjugate was administered at a dose of 400 praoles per injection on days 3, 5, 7, 9, 11, and 13, (SMG-I (IX)), or at a dose of 800 pmoles per injection administered at days 3, 5, 7, * ⁇ >, 1 1, 13, 15 and 17, (SMG-I (2X ).
  • Figure 14A Survival. The long term surviving mice (> 40 days) had no evidence of tumor.
  • Statistical analysis SMG-I (I X) versus Figure 14B. Tumor size.
  • FIG. 15 PSMA aptamer-SMG-1 siRNA rejection of PSMA-expr ⁇ ssing, but not parental, CT26 tumor cells - Tumor size at day of sacrifice. Mice were sacrificed at day 19 when tumors in the PBS group reached maximum allowable size (12 mm diameter). Only the PSMA- CT26 tumors in mice treated with PSMA-SMG-I siRNA have shown consistently diminished growth compared to contralateral ⁇ implanted parental CT26 tumors; in this experiment in 7 out of 8 mice the PSMA-CT26 tumors were cither smaller than the parental CT26 tumors or completely regressed.
  • genes, gene names, and gene products disclosed herein are intended to correspond to homologs from any species for which the compositions and methods disclosed herein are applicable, Thus, the terms include, but are not limited to genes and gene products from humans and mice. It is understood that when a gene or gene product from a particular species is disclosed, this disclosure is intended to be exemplary only, arid is not to be interpreted as a limitation unless the context in which it appears clearly indicates. Thus, for example, for the genes disclosed herein, which in some embodiments relate to mammalian nucleic acid and amino acid sequences are intended to encompass homologous and/or orthologous genes and gene products from other animals including, but not limited to other mammals, fish, amphibians, reptiles, and birds. In preferred embodiments, the genes or nucleic acid sequences are human.
  • a "target cell” or “recipient cell” refers to an individual cell or cell which is desired to be, or has been, a recipient of exogenous nucleic acid molecules
  • polynucleotides and/or proteins are also intended to include progeny of a single cell.
  • nucleic acid aptamcr a nucleic acid molecule that binds specifically to a target molecule wherein the nucleic acid molecule has sequence that comprises a sequence recognized by the target molecule in its natural setting.
  • an aptam ⁇ r can be a nucleic acid molecule that binds to a target molecule wherein the target molecule does not naturally bind to a nucleic acid.
  • the target molecule can be any molecule of interest,
  • the aplamer can be used to bind to a ligand-binding domain of a protein, thereby preventing interaction of the naturally occurring ligand with the protein. This is a non-limiting example and those in the art will recognize that other embodiments can be readily generated using techniques generally known in the art (see, e.g., Gold ei ⁇ /., Annu. Rev. Biochem. 64:763. 1995; Brody and Gold, J.
  • multi-domain molecules refers to the different variations of the therapeutic molecules that comprise a domain which specifically targets or delivers the molecule to a desired ceil or in vivo locale and a second domain which modulates expression or function of the nonsense mediated decay pathway or molecules associated with these pathways,
  • the multi-domain molecule can comprise at least one aptamer conjugated, linked, fused, etc., to an oligonucleotide such as a siRNA, which modulates the function of the NMD pathway and/or the expression and function of a molecule associated with the NMD pathway.
  • aptamer-oligonucl ⁇ otide refers to the compositions described herein wherein at least one aptamer is linked or conjugated to at least one antisense oligonucleotide. Combinations of more than one aptamer and oligonucleotides, with more than one specificity, arc included.
  • oligonucleotide specific for refers to an oligonucleotide having a sequence (i) capable of forming a stable complex with a portion of the targeted gene, or (if) capable of forming a stable duplex with a portion of a mRNA transcript of the targeted gene.
  • oligonucleotide is meant to encompass all forms or desired RNA, RNA/DNA molecules which modulate gene expression and /or function, and includes without limitation: “siRNA,” “shRNA” “antisense oligonucleotide” etc.
  • the term also includes linear or circular oligomers of natural and/or modified monomers or linkages, including dcoxyribonuclcosides, ribonucleosides, substituted and alpha-anomeric forms thereof, peptide nucleic acids (PNA), locked nucleic acids (LNA), phosphorothioate, methylphosphonate, and the like.
  • Oligonucleotides are capable of specifically binding to a target polynucleotide by way of a regular pattern of monomer-to-monomer interactions, such as Watson-Crick type of base pairing, Ho ⁇ gst ⁇ en or reverse Hoogsteen types of base pairing, or the like.
  • the aptamer-oligonucleotidc may be "chimeric,” that is, composed of different regions.
  • "chimeric" compounds are aptamer-oiigonucleotides, which contain two or more chemical regions, for example, DNA region(s), RNA region(s), PNA region(s) etc. Each chemical region is made up of at least one monomer unit, i.e., a nucleotide in the case of an oligonucleotide compound.
  • These oligonucleotides typically comprise at least one region wherein the oligonucleotide is modified in order to exhibit one or more desired properties.
  • the desired properties of the oligonucleotide include, but are not limited, for example, to increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity for the target nucleic acid. Different regions of the oligonucleotide may therefore have different properties.
  • the chimeric oligonucleotides of the present invention can be formed as mixed structures of two or more oligonucleotides, modified oligonucleotides, oligonucleosid.es and/or oligonucleotide analogs as described above.
  • the oligonucleotide can be composed of regions that can be linked in "register,” that is, when the monomers are linked consecutively, as in native DNA, or linked via spacers.
  • the spacers are intended to constitute a covalent "bridge” between the regions and have in preferred cases a length not exceeding about 100 carbon atoms.
  • the spacers may cany different functionalities, for example, having positive or negative charge, carry special nucleic acid binding properties (intercalators, groove binders, toxins, iluorophors etc.), being lipophilic, inducing special secondary stnictures like, for example, alanine containing peptides that induce alpha-helices.
  • oligonucleotide specific for refers to an oligonucleotide having a sequence (i) capable of forming a stable complex with a portion of the targeted gene, or (H) capable of forming a stable duplex with a portion of a mRNA transcript of the targeted gene.
  • the term "monomers” typically indicates monomers linked by phosphodiesler bonds or analogs thereof to form oligonucleotides ranging in size from a few monomcric units, e.g., from about 3-4, to about several hundreds of monomelic units.
  • Analogs of phosphodiester linkages include: phosphorolhioate, phosphorodithioate, melhylphosphornates, phosphoroselenoate, phosphoramidate, and the like, as more fully described below.
  • nuclcobase covers naturally occurring nuclcobases as well as non-naturally occurring nucleobases. It should be clear to the person skilled in the art that various nucleobases which previously have been considered “non-naturally occurring” have subsequently been found in nature. Thus, “nucleobase” includes not only the known purine and pyrimidine heterocycles, but also heterocyclic analogues and tautomers thereof.
  • nucleobases are adenine, guanine, thymine, cytosi ⁇ , uracil, purine, xanthine, diaminopurine, 8-oxo-N 6 -methyladenine, 7-deazaxanthin ⁇ , 7-deazaguanine, N 4 , Nf- elhanocytosin. N°,N b -et3iano-2,6-diarninopurme.
  • nucleobase is intended to cover every and all of these examples as well as analogues and tautomers thereof.
  • nucieobases are adenine, guanine, thymine, cytosine, arid uracil, which are considered as the naturally occurring nucieobases in relation to therapeutic and diagnostic application in humans.
  • nucleoside includes the natural nucleosides, including 2'-deoxy and 2'-hydroxyl forms, e.g., as described in Kornberg and Baker, DNA Replication, 2nd Ed.
  • nucleosides in reference to nucleosides includes synthetic nucleosides having modified base moieties and/or modified sugar moieties, e.g., described generally by Scheil, Nucleotide Analogs, John Wiley, New York, 1980; Freier & Altmann, Nucl. Acid Res., 1997, 25(22), 4429- 4443, Toulme, J J., Nature Biotechnology 19: 17-18 (2001); Manoharan M,, Biochemica el Biophysica Acta 1489:1 17-139(1999); Freier S.,M, Nucleic Acid Research, 25:4429-4443 (1997), Uhlman, E., Drug Discovei ⁇ & Development.. 3: 203-213 (2000), Hcrdcwin P., Anti sense ⁇ Nucleic Acid Drug Dev., 10:297-310 (2000), ⁇ ; 2'-O, 3 * -C-linked [3.2.0]
  • bicycloarabinonucleosides see e.g. N. K Christienscn., el al. J. Am. Chem. Soc. h 120: 5458-5463 (1998).
  • Such analogs include synthetic nucleosides designed to enhance binding properties, e.g., duplex or triplex stability, specificity, or the like.
  • the term "gene” means the gene and all currently known variants thereof and any further variants which may be elucidated.
  • variant of polypeptides refers to an amino acid sequence that is altered by one or more amino acid residues.
  • the variant may have "conservative” changes, wherein a substituted amino acid has similar structural or chemical properties ⁇ e.g., replacement of leucine with isolcucine). More rarely, a variant may have "nonconservative" changes (e.g. * replacement of glycine with tryptophan).
  • Analogous minor variations may also include amino acid deletions or insertions, or both.
  • Guidance in determining which amino acid residues may be substituted, inserted, or deleted without abolishing biological activity may be found using computer programs well known in the art, for example, LASERGENE software (DNASTAR).
  • variant when used in the context of a polynucleotide sequence, may encompass a polynucleotide sequence related to a wild type gene. This definition may also include, for example, “allelic,” “splice,” “species,” or “polymorphic” variants. A splice variant may have significant identity to a reference molecule, but will generally have a greater or lesser
  • Species variants are polynucleotide sequences that vary from one species to another. Of particular utility in the invention arc variants of wild type target gene products. Variants may result from at least one mutation in the nucleic acid sequence and may result in altered niRNAs or in polypeptides whose structure or function may or may not be altered. Any given natural or recombinant gene may have none, one, or many allelic forms. Common mutational changes that give rise to variants are generally ascribed to natural deletions, additions, or substitutions of nucleotides. Each of these types of changes may occur alone, or in combination with the others, one or more times in a given sequence.
  • polymorphic variant is a variation in the polynucleotide sequence of a particular gene between individuals of a given species. Polymorphic variants also may encompass "single nucleotide polymorphisms" (SNPs,) or single base mutations in which the polynucleotide sequence varies by one base. The presence of SNPs may be indicative of, for example, a certain population with a propensity for a disease state, that is susceptibility versus resistance.
  • SNPs single nucleotide polymorphisms
  • mRNA means the presently known mRNA transcript(s) of a targeted gene, and any further transcripts which may be elucidated.
  • RNA molecules any foreign RNA molecule which is useful from a therapeutic, diagnostic, or other viewpoint.
  • Such molecules include anlisense RNA molecules, decoy RNA molecules, enzymatic RNA, therapeutic editing RNA and agonist and antagonist RNA,
  • antisense RNA is meant a non-enzymatic RNA molecule that binds to another RNA (target RNA) by means of RNA-RNA interactions and alters the activity of the target RNA (Eguchi et ai, 1991 Annu. Rev. Biochem. 60, 631-652),
  • RNA interference "RNAi” is mediated by double stranded RNA (dsRNA) molecules that have sequence-specific homology to their "target” nucleic acid sequences (Caplcn, N. J., et oL. P roc. Nad, Acad, Sci. USA 98:9742-9747 (2001)).
  • dsRNA double stranded RNA
  • the mediators of RNA-dependent gene silencing are 21 -25 nucleotide "small interfering" RNA duplexes (si R NAs),
  • the siRNAs are derived from the processing of dsRNA by an RNasc enzyme known as Dicer (Bernstein, E., et al, Nature 409:363-366 (2001)), siRNA duplex products are recruited into a multi-protein si RNA complex termed RISC (RNA Induced Silencing Complex).
  • a R ISC is then believed to be guided to a target nucleic acid (suitably mRNA), where the siRNA duplex interacts in a sequence-specific way to mediate cleavage in a catalytic fashion (Bernstein, E., ei al. Nature 409:363-366 (2001): Boutla. A., et al. Curr. Biol.
  • a target nucleic acid suitably mRNA
  • Small interfering RNAs that can be used in accordance with the present invention can be synthesized and used according to procedures that are well known in the art and that will be familiar to the ordinarily skilled artisan, Small interfering RNAs for use in the methods of the present invention suitably comprise between about 0 to about 50 nucleotides (nt).
  • nt nucleotides
  • siRNAs can comprise about 5 to about 40 nt, about 5 to about 30 nt, about 10 to about 30 nt, about 15 to about 25 nt, or about 20-25 nucleotides.
  • RNAi is facilitated by using computer programs that automatically align nucleic acid sequences and indicate regions of identity or homology. Such programs are used to compare nucleic acid sequences obtained, for example, by searching databases such as GcnBank or by sequencing PCR products, Comparison of nucleic acid sequences from a range of species allows the selection of nucleic acid sequences that display an appropriate degree of identity between species, In the case of genes that have not been sequenced, Southern blots are performed to allow a determination of the degree of identity between genes in target species and other species. By performing Southern blots at varying degrees of stringency, as is well known in the art, it is possible to obtain an approximate measure of identity.
  • RNAi that exhibit a high degree of complementarity to target nucleic acid sequences in a subject to be controlled and a lower degree of complementarity to corresponding nucleic acid sequences in other species.
  • One skilled in the art will realize that there is considerable latitude in selecting appropriate regions of genes for use in the present invention.
  • enzymatic RNA an RNA molecule with enzymatic activity (Cech, 1988 J. American. Med, Assoc, 260, 3030-3035).
  • Enzymatic nucleic acids ribozymes act by first binding to a target RNA. Such binding occurs through the target binding portion of a enzymatic nucleic acid which is held in close proximity to an enzymatic portion of the molecule that acts to cleave the target RNA.
  • the enzymatic nucleic acid first recognizes and then binds a target RNA through base-pairing, and once bound to the correct site, acts enzymatically to cut the target RNA.
  • decoy RNA is meant an RNA molecule that mimics the natural binding domain for a ligand.
  • the decoy RNA therefore competes with natural binding target for the binding of a specific ligand.
  • TAR HIV trans-activation response
  • TAR HIV trans-activation response
  • RNA can act as a "decoy” and efficiently binds HIV tat protein, thereby preventing it from binding to TAR sequences encoded in the HIV RNA (Sullenger et a!.. 1990, Cell, 63, 601 -608).
  • This is meant to be a specific example. Those in the art will recognize that this is but one example, and other embodiments can be readily generated using techniques generally known in the art.
  • complementary means that two sequences are complementary when the sequence of one can bind to the sequence of the other in an anti-parallel sense wherein the 3 '-end of each sequence binds to the 5'-end of the other sequence and each A, T(Ii), G, and C of one sequence is then aligned with a T(U), A, C, and G, respectively, of the other sequence.
  • the complementary sequence of the oligonucleotide has at least 80% or 90%, preferably 95%, most preferably 100%, complementarity to a defined sequence.
  • alleles or variants thereof can be identified.
  • a BLAST program also can be employed to assess such sequence identity.
  • complementary sequence as it refers to a polynucleotide sequence, relates to the base sequence in another nucleic acid molecule by the base-pairing rules. More particularly, the term or like term refers to the hybridization or base pairing between nucleotides or nucleic acids, such as, for instance, between the two strands of a double stranded DNA molecule or between an oligonucleotide primer and a primer binding site on a single stranded nucleic acid to be sequenccd or amplified.
  • Complementary nucleotides are, generally, A and T (or A and U), or C? and G.
  • Two single stranded RNA or DNA molecules are said to be substantially complementary when the nucleotides of one strand, optimally aligned and compared and with appropriate nucleotide insertions or deletions, pair with at least about 95% of the nucleotides of the other strand, usually at least about 98%, and more preferably from about 99 % to about 100%.
  • Complementary polynucleotide sequences can be identified by a variety of approaches including use of well-known computer algorithms and software, for example the BLAST program,
  • the terra "stability" in reference to duplex or triplex formation generally designates how tightly an antis ⁇ nse oligonucleotide binds to its intended target sequence; more particularly, "stability" designates the ⁇ JQQ energy of formation of the duplex or triplex under physiological conditions. Melting temperature under a standard set of conditions, e.g., as described below, is a convenient measure of duplex and/or triplex stability.
  • oligonucleotides of the invention are selected that have melting temperatures of at least 45°C when measured in 100 rtiM NaCL 0.1 niM EDTA and 10 rnM phosphate buffer aqueous solution, pH 7.0 at a strand concentration of both the oligonucleotide and the target nucleic acid of 1.5 ⁇ M.
  • duplex or triplex formation will be substantially favored over the state in which the antigen and its target are dissociated.
  • a stable duplex or triplex may in some embodiments include mismatches between base pairs and/or among base triplets in the case of triplexes.
  • modified oligonucleotides, e.g. comprising LNA units, of the invention fo ⁇ n perfectly matched duplexes and/or triplexes with their target nucleic acids.
  • the term "Thermal Melting Point (Tm)" refers to the temperature, under defined ionic strength, pH, and nucleic acid concentration, at which 50% of the oligonucleotides complementary to the target sequence hybridize to the target sequence at equilibrium. As the target sequences are generally present in excess, at Tm, 50% of the oligonucleotides arc occupied at equilibrium).
  • stringent conditions will be those in which the salt concentration is at least about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 3O 0 C. for short oligonucleotides (e.g., 10 to 50 nucleotide). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamid ⁇ .
  • stringent conditions refers to conditions under which an oligonucleotide will hybridize to its target subsequence, but with only insubstantial hybridization to other sequences or to other sequences such that the difference may be identified. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. Generally, stringent conditions are selected to be
  • Tm thermal melting point
  • the terra "target nucleic acid” refers to a nucleic acid (often derived from a biological sample), to which the oligonucleotide is designed to specifically hybridize. It is either the presence or absence of the target nucleic acid that is to be delected, or the amount of the target nucleic acid that is to be quantified.
  • the target nucleic acid has a sequence that is
  • target nucleic acid may refer to the specific subsequence of a larger nucleic acid to which the oligonucleotide is directed or to the overall sequence (e.g., gene or mRNA) whose expression level it is desired to detect. The difference in usage will be apparent from context.
  • Target molecule includes any macromolecule, including protein, carbohydrate, enzyme, polysaccharide, glycoprotein, receptor, antigen, antibody, growth factor; or il may be any small organic molecule including a hormone, substrate, metabolite, cofactor, inhibitor, drug, dye, nutrient, pesticide, peptide; or it may be an inorganic molecule including a metal, metal ion, metal oxide, and metal complex; it may also be an entire organism including a bacterium, virus, and single-cell eukaryote such as a protozoon.
  • modulate it is meant that any of the mentioned activities, arc, e.g., increased, enhanced, increased, agonized (acts as an agonist), promoted, decreased, reduced, suppressed blocked, or antagonized (acts as an agonist). Modulation can increase activity more than 1-fold, 2 -fold, 3-fold, 5-fold, 10-fold. 100-fold, etc., over baseline values. Modulation can also decrease its activity below baseline values. Modulation can also normalize an activity to a baseline value.
  • a "pharmaceutically acceptable” component/carrier etc is one that is suitable for use with humans and/or animals without undue adverse side effects (such as toxicity, irritation, and allergic response) commensurate with a reasonable benefit/risk ratio,
  • safety and effective amount refers to the quantity of a component which is sufficient to yield a desired therapeutic response without undue adverse side effects (such as toxicity, irritation, or allergic response) commensurate with a reasonable benefit/risk ratio when used in the manner of this invention.
  • therapeutically effective amount is meant an amount of a compound of the present invention effective Io yield the desired therapeutic response. For example, an amount effective to delay the growth of or to cause a cancer, either a sarcoma or lymphoma, or to shrink the cancer or prevent metastasis.
  • the specific safe and effective amount or therapeutically effective amount will vary with such factors as the particular condition being treated, the physical condition of the patient, the type of mammal or animal being treated, the duration of the treatment, the nature of concurrent therapy (if any), and the specific formulations employed and the structure of the compounds or its derivatives.
  • a "pharmaceutical salt” include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids.
  • the salts are made using an organic or inorganic acid.
  • These preferred acid salts are chlorides, bromides, sulfates, nitrates, phosphates, sulfonates, formates, tartrates, maleates, malates, citrates, benzoates, salicylates, ascorbates, and the like.
  • the most preferred salt is the hydrochloride salt.
  • Diagnostic or “diagnosed” means identifying the presence or nature of a pathologic condition. Diagnostic methods differ in their sensitivity arid specificity, The "sensitivity” of a diagnostic assay is the percentage of diseased individuals who test positive (percent of "true positives”). Diseased individuals riot detected by the assay are “false negatives," Subjects who are not diseased and who test negative in the assay, are termed “true negatives,” The
  • patient or “individual” are used interchangeably herein, and refers to a mammalian subject to be treated, with human patients being preferred.
  • the methods of the invention find use in experimental animals, in veterinary application, and in the development of animal models for disease, including, but not limited to, rodents including mice, rats, and hamsters; and primates.
  • Treatment is an intervention performed with the intention of preventing the development or altering the pathology or symptoms of a disorder. Accordingly, “treatment” refers to both therapeutic treatment and prophylactic or preventative measures. “Treatment” may also be specified as palliative care. Those in need of treatment include those already with the disorder as well as those in which the disorder is Io be prevented. In tumor (e.g., cancer) treatment, a therapeutic agent may directly decrease the pathology of tumor cells, or render the tumor cells more susceptible to treatment by other therapeutic agents, e.g., radiation and/or chemotherapy.
  • other therapeutic agents e.g., radiation and/or chemotherapy.
  • treating 1 " or “treatment 1 " of a state, disorder or condition includes: (1) preventing or delaying the appearance of clinical symptoms of the state, disorder or condition developing in a human or oilier mammal that may be afflicted with or predisposed to the state, disorder or condition but does not yet experience or display clinical or subclinical symptoms of the state, disorder or condition; (2) inhibiting the state, disorder or condition, i.e..
  • the benefit to an individual to be treated is either statistically significant or at least perceptible to the patient or to the physician.
  • Disseminated metastatic disease is the primary cause of death among cancer patients, Cancer vaccination stimulates a systemic immune response against judiciously chosen tumor antigens expressed in the tumor cells that seeks out and destroys the disseminated tumor lesions.
  • TRAs tumor rejection antigens
  • the development of effective cancer vaccines will require the identification of potent and broadly expressed tumor rejection antigens (TRAs) (Gilboa, E. Immunity 1 1, 263-270 ( 1999); Novellino, L., et al. Cancer Immunol, Immunother. 54, 187-207 (2005); Schietingcr, A,, ct al. Semin. Immunol. 20, 276-285 (2008)) as well as effective adjuvants to stimulate a robust and durable immune response (Gilboa, E. Nature Rev. Cancer 4, 401-411 (2004); Melief, C. J. Immunity 29, 372-383 (2008); Pardoll, D. M. Nature Rev. Immunol. 2, 227-238 (2002)).
  • Embodiments of the invention comprise expressing new, and hence potent, antigens in tumor cells in situ, How to express new antigens in the disseminated tumor lesions, but not in normal tissue, have heretofore precluded the development of such strategics.
  • Nonsense mediated decay prevents the expression of aberrant products in the cell.
  • inhibition of NMD in tumor cells leads to the expression of novel antigens and enhancement of the immimogenicity of tumor cells, leading to tumor rejection by the immune response.
  • Delivery of siRNA targeted to NMD pathways in vivo can be used to inhibit NMD.
  • non-targeted delivery of siRNA in vivo is not clinically practical because of cost consideration and anticipated toxicity. Targeting si RNA to the appropriate cells, tumor cells in this instance, would solve the problem.
  • antibodies are the obvious choice for targeting ligands. However, since antibodies are cell based products, and hence pose significant cost, manufacturing, and regulatory challenges.
  • oligonucleotide -based aptamers - is a novel platform technology that can be used to develop improved methods of delivering therapeutic cargo such as small MVV drugs, toxins, siRNAs, as well as therapeutic aptamers, to cells hi vivo.
  • therapeutic cargo such as small MVV drugs, toxins, siRNAs, as well as therapeutic aptamers, to cells hi vivo.
  • compositions described herein provide novel methods of in vivo drug targeting that offers potential advantages over the use of antibodies and protein-based therapeutic agents as targeting ligands.
  • the invention provides compositions and methods for inhibiting nonsense- mediated raRNA decay (NMD) and/or a component of the NMD pathway in a cell.
  • NMD nonsense- mediated raRNA decay
  • Embodiments of the invention are directed to a clinically useful approach to express new antigens in the disseminated tumor lesions of cancer patients by targeted inhibition of nonsense mediated decay (NMD) in the tumor cells.
  • NMD is an evolutionary conserved mRNA surveillance pathway in eukaryotic cells that detects and eliminates mRNAs harboring premature termination codons (PTCs).
  • PTCs premature termination codons
  • the targeted NMD inhibitory agent comprises at least a single chemically synthesized oligonucleotide molecule.
  • a composition for inducing novel antigens in abnormal cells comprises a multi-domain molecule having at least one target specific domain and at least one domain, which modulates expression and function of molecules associated with nonsense mediated decay pathways.
  • the multi-domain molecule comprises at least one target specific domain and at least two domains which modulate expression and function of one or more molecules associated with nonsense mediated decay pathways.
  • examples of such molecules comprise: RENTl, RENT2, eIF4A, UPFl , IJPF2, UPF3B, RNPSl, Y14, MAGOH, NMDL SMG, or combinations thereof.
  • the multi-domain molecule comprises at least two target specific domains and at least one domain which modulates expression and function of one or more molecules associated with nonsense mediated decay pathways.
  • the target specific domains comprise specificities for similar target molecules, different target molecules, or combinations thereof.
  • the domains which modulate expression and function of one or more molecules associated with nonsense mediated decay pathways modulate the expression and function of similar targeted molecules, different targeted molecules or combinations thereof.
  • the target specific domains are specific for target cell molecules, the target cell comprising: a tumor cell, an infected cell, a tissue specific cell, an adipocyte, a stem cell, an immune cell, an organ specific ceil or a transformed cell.
  • NMD Nonsense mediated (mRNA) decay
  • Figure 1 is a schematic representation showing the mechanism by which the NMD process prevents the accumulation of PTC containing mRN As in eukaryotic cells.
  • EJC exon-junction complex
  • An NMD complex consisting of several factors including Upfl, Upf2 and lJp3 is then assembled on each EJC as shown in Panel B.
  • SMGl which phospliorylatcs Upfl, and Upfl arc the two key rate limiting factors in the formation of the complex.
  • the EJCz 1 NMD complex When the mRNA undergoes the first round of translation, called the "pioneer translation", the EJCz 1 NMD complex is removed, presumably as a result of the translational machinery moving through the region, thereby rendering the mRNA stable and competent for additional rounds of translation. If a PTC is present in an exon (other than the last exon), for example as a result of an inframe nonsense mutation, the EJC/NMD complexes downstream to the PTC are not removed from the mRNA. The attached NMD complex then triggers the degradation of the mRNA,
  • a composition comprising oligonucleotides directed against NMD-sp ⁇ cific factors inhibit NMD in tumor cells (see, for example, Figure 1 ).
  • the siRNAs targeted to the tumor by conjugation to aptamer-bas ⁇ d ligands. Targeting the si RNAs to tumor cells is a preferred method for the therapeutic use of this approach since upregulation of new products in nontransformed cells could expose normal tissue to immune destruction creating an "autoimmune inferno".
  • 10082 J NMD-mediated degradation of mRNA is not limited to instances of mutations or recombinations generating PTCs.
  • Error-free raRNAs containing short 5' open reading frames are not limited to instances of mutations or recombinations generating PTCs.
  • mRNAs which are regulated by stop codon read-through, leaky scanning for translation initiation, or regulated framesl ⁇ fting or mRN A can be also recognized by NMD,
  • Physiological roles 0/A 1 MD It was initially thought that the main role of NMD was to maintain the proteome integrity of the cell by eliminating transcripts with nonsense mutations generating PTCs yielding truncated products. Indeed, over 30% of genetic disorders are caused by PTC. In several instances the severity of the disease, e.g. ⁇ -thalass ⁇ mia, correlates with the NMD-controllcd degradation of the mutant mRN A. Yet, nonsense mutations generating PTCs are rare events and it is unlikely that the NMD system has evolved to counter their potential deleterious effects. There is in fact accumulating evidence that the main and physiological role of the NMD is to regulate normal gene expression.
  • NMD Newcastle disease virus
  • the efficiency and accuracy of splicing is notoriously imperfect, Such transcripts will often contain PTCs and hence become targets for NMD elimination.
  • NMD is also responsible for the elimination of transcripts encoding nonproductively rearranged T cell receptors and immunoglobulin chains, A significant proportion of gene products (>15%) thai are upregulated when NMD is inhibited, such as by targeting Upfl with siRNA are involved in amino acid biosynthesis and transcription factors which coordinate cellular responses to starvation.
  • NMD is also implicated in several instances of products autoregulating alternative splicing (e.g., serine-arginine (SR)- rich proteins and hnRNP splicing factors such as SC35, calpain, CDC-like kinases, biosynthesis of selenoproteins, and telomere synthesis.
  • SR serine-arginine
  • hnRNP splicing factors such as SC35, calpain, CDC-like kinases, biosynthesis of selenoproteins, and telomere synthesis.
  • a method of inducing or enhancing tumor antigenicity comprises a composition having an aptamcr specific for a target cell conjugated to an agent which inhibits NMD in tumor cells, for example, siRN As specific for key NMD factors.
  • Upfl also promotes the replication-dependent decay of histone mRNA which is required for cell cycle progression.
  • SMGl is a kinase which also phosphorylates and inactivates p53; siRN A inhibition of SMG- 1 in LJ20S cells results in the accumulation of dsDNA break and activation of ATM- or ATR -mediated checkpoint responses, Both Upfl and SMGl were implicated in telomere maintenance by facilitating the binding of telomere repeat- containing RNA (TERRA) Io telomeres,
  • Cancer cells accumulate elevated level of PTC containing NMD mRNA substrates, About 15% of cancers exhibit defects in DNA mismatch repair (MMR) often manifested as mitellite instability (MSI). Such detects affecting many products, including products associated with tumor progression such as TGF ⁇ Rii, APAF-I, IGFIIR, BAX, PTEN, RHAMM, give rise to framcshift mutation ending in PTCs.
  • MMR DNA mismatch repair
  • MSI miratellite instability
  • Such PTC-containing transcripts are under NMD control whereby Upfl siRNA mediated inhibition of NMD in a human colorectal cancer cell line exhibiting an MSI phenotype stabilized the framcshiftcd mutant transcripts.
  • Such products could provide a source of tumor-specific antigenic determinants downstream the recombination site.
  • increased immune infiltrate are seen in tumors with MlS phenotype would correlate with the levels of Upfl in the tumors.
  • Inhibiting NMD further augments the production
  • ⁇ ptamer mediated targeting ofsiRNA Targeting the siRNAs to tumor cells is a preferred embodiment.
  • the methods utilize the following approach: Q). Upregulation of new products in nontransformed cells would expose normal tissue to immune destruction creating an "autoimmune inferno.” (ii). NMD is a physiologically important process regulating various house-keeping functions of somatic cells and the key factors of the NMD process, SMGl and Upfl , play also important roles in maintaining genome stability and cells survival Inhibiting NMD in somatic cells would be, deleterious, (iii). Targeting the oligonucleotide- based aptamer- oligonucleotidcs agent to tumor cells will reduce the cost of treatment and the risk of adverse effects associated with the non-specific stimulatory properties of nucleic acids.
  • Monoclonal antibodies have been used as ligands with engineered specificity to target drugs, toxins as well as siRNAs to cells.
  • a major limitation of using antibodies in therapeutic settings is limited, and at best uncertain, access to this class of biologicals. The reason is that antibodies arc cell-based products posing significant cost, manufacturing and regulator ⁇ ' challenges.
  • clinical-grade reagents are almost exclusively developed and provided by companies on a selective basis and under strict contractual agreement.
  • aptamers are high affinity single stranded nucleic acid ligands which can be isolated trough a combinatorial chemistry process known as SELEX.
  • Aptamers with nuclease-resistant backbone can be generated against most targets, proteins as well as small molecules, and exhibit remarkable affinity and specificity to their targets comparable to and often exceeding that of antibodies.
  • the 25-40 nt long ap tamers can be synthesized chemically, Consequently, manufacture of clinical grade aptamers, including aplamer-oligonucieotides fusion ODNs, is relatively cost effective, and the regulatory approval process significantly simpler.
  • Figure 2 shows how aptamers can be used to target siRNAs to tumor cells.
  • an aptatner is inclusive of one or more aptamers that may have the same specificity for a target molecule, or the aptamers are specific for different targets. Thus, when using the term “aptatner” the term applies to one or a plurality of aptamers linked or conjugated together and can each be specific for different target antigens.
  • the gene silencing agent (the RNAi) is targeted to the appropriate cells in vivo using nuclease-resistant oligonucleotide -based aptamers.
  • Targeting of polynucleotides without limitation, any one or more components of a nonsense mediated decay- pathway.
  • the display of new antigens or an increase in antigens that can be recognized by the immune system as abnormal or foreign results in the destruction of that cell.
  • a composition comprising a targeting agent and a gene silencing agent down-regulate or abrogate nonsense mediated decay pathways.
  • the gene silencing agent is an RNAi (siRNA/sliRNA).
  • siRNAs to SMG-I, Upf2 and UpO were characterized and stably transduced CT26 tumor cells with a ientivirus-based vector (LV) expressing the siRNAs from a tct-lnducible U ⁇ promoter. Having confirmed that doxycyclinc treatment induced siRNA expression and NMD inhibition in the culture cells, measured as downregulation of the corresponding mRNA by semiquantitative RT-PCR and stabilization of a PTC-containing mRNA expressed from an NMD reporter plasmid. the tumor cells were implanted in mice and tumor growth was monitored in the presence or absence of doxy eye line. siRNA inhibition of SMG-I or U ⁇ f2 led to an almost complete inhibition of tumor growth.
  • LV ientivirus-based vector
  • the siRNA, antisense oligonucleotides are directed to factors associated with the NM D pathway comprising at least one of: RENTl, RENT2, eIF4A, UPFl, UPF2, ⁇ JPF3B, RNPSl, Y14, MAGOH, NMDl or SMG.
  • the aptam ⁇ r-oligonucleotid ⁇ molecule can be administered in conjunction with one or more agents which inhibit NMD.
  • agents which inhibit NMD for example, use of pharmacological agents that inhibit protein translation. Examples of such drugs are described in Noensic and Dietz ((2001) Nature Biotech 19: 434-439), the contents of which are incorporated herein by reference. This approach is based upon the finding that NMD is generally inhibited by agent that block or inhibit protein translation. Examples of such agents include emetine, anisomycin, cycloheximide, pactamycin, puromycin, gentamicin, neomycin, and paromomycin.
  • a nucleic acid is associated with the aptamers.
  • the nucleic acid can be selected from a variety of DN A and RNA based nucleic acids, including fragments and analogues of these.
  • a variety of genes for treatment of various conditions have been described, and coding sequences for specific genes of interest can be retrieved from DNA sequence databanks, such as Gen Bank or EMBL.
  • polynucleotides for treatment of viral, malignant and inflammatory diseases and conditions such as, cystic fibrosis, adenosine deaminase deficiency and AIDS, have been described.
  • Treatment of cancers by administration of tumor suppressor genes such as APC, DPC4, NF-I, NF-2, MTSl, RB, p53, WTl, BRCAl, BRCA2 and VHL, are contemplated.
  • nucleic acids for treatment of an indicated conditions include: HLA-B7, tumors, colorectal carcinoma, melanoma; 1L-2, cancers, especially breast cancer, lung cancer, and tumors; IL-4, cancer; TNF, cancer; IGF-I antisense, brain tumors; IFN,
  • the polynucleotide can be an antisense DNA oligonucleotide composed of sequences complementary to its target, usually a messenger RNA (mRNA) or an mRN A precursor.
  • mRNA messenger RNA
  • mRN A precursor a messenger RNA (mRNA) or an mRN A precursor.
  • the mRNA contains genetic information in the functional, or sense, orientation and binding of the antisense oligonucleotide inactivates the intended mRNA and prevents its translation into protein.
  • antisense molecules are determined based on biochemical experiments showing that proteins are translated from specific RNAs and once the sequence of the RNA is known, an anlisense molecule that will bind Io it through complementary Watson-Crick base pairs can be designed.
  • Such antisense molecules typically contain between 10-30 base pairs, more preferably between 10-25, and most preferably between 15-20.
  • the antisense oligonucleotide can be modified for improved resistance to nuclease hydrolysis, and such analogues include phosphorothioate, methylphosphonat ⁇ , phosphodiester and p-elhoxy oligonucleotides (WO 97/07784).
  • the aptamer can be specific for any type of products, for example, tumor antigens, cell adhesion molecules, such as for example, integrins, glycosylated proteins, etc.
  • cell adhesion molecules such as integrins
  • RGD is an alternative to aptamer - it targets "cargo" to inflammed endothelial cells.
  • jOlOli Other examples comprise: stromal derived factor 1 (SDF-I ), MCP-I, MIP- l ⁇ , MlP- l ⁇ , RANTES, exotaxin IL-8, C3a, P-selcctin, E-selcctin, LFA-I, VLA-4, VLA-5, CD44, MMP activation, VEGF, EGF, PDGF, VCAM, ECAM, G-CSF, GM-CSF, SCF, EPO, tenascin, MAdCAM-I, o:4 integrins, ⁇ 5 integrins, beta defensins 3 and 4.
  • SDF-I stromal derived factor 1
  • MCP-I MCP-I
  • MIP- l ⁇ MlP- l ⁇
  • RANTES exotaxin IL-8
  • C3a P-selcctin
  • E-selcctin LFA-
  • the target molecule can be one that binds to, for example, an extracellular domain of a growth factor receptor.
  • exemplary receptors include the c-erbB-2 protein product of the H£R2/n ⁇ u oncogene, epidermal growth factor (EGF) receptor, basic fibroblast growth receptor (basic FGF) receptor and vascular endothelial growth factor receptor, E-, L- and P-selectin receptors, folate receptor, CD4 receptor, CD 19 receptor, ⁇ , ⁇ - integrin receptors and chemokine receptors.
  • the aptaniers may also be conjugated to transporter proteins to increase the transportation of the oligonucleotides specific for NMD factors across membranes e.g. blood brain barrier, intestines, etc.
  • a "chemotherapeutic agent" which can also be the cargo moiety for treatment of a tumor is a chemical compound useful in the treatment of cancer
  • chemotherapeulic agents include alkylating agents such as thiotepa and cyelosphosphamide (CYTOXANTM); alkyl sulfonates such as busulfan, improsulfan and piposulfan: aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and m ⁇ thylani ⁇ lamin ⁇ s including altretamiiie, tricthylcncmclaminc, trictylenephosphoraniidc, tri
  • cholophosphamide estramustine, ifosfamide, mechlorethamin ⁇ , mechiorethamine oxide hydrochloride, nielphalan, novernbichin, pheneslerine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustm ⁇ , lomustine, nimustine, ranimustinc: antibiotics such as aclacinomysins, actinomycin, aulhramycin, azascrinc, bleomycins, cactinomycin, calicheamicin, carabicin, carnomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, dctorubicin, ⁇ -diazo-S-oxo-L-norlcucine, doxorubicin, epirubicin, esorubic
  • paclitaxel TAXOL®, Bristol-Myers Squibb Oncology, Princeton, NJ.
  • docetax ⁇ l TAXOL®, Bristol-Myers Squibb Oncology, Princeton, NJ.
  • TXOTERE® Rh ⁇ n ⁇ -Poulcnc Rorer, Antony, France
  • chlorambucil gemcitabinc
  • 6- thioguanine mercaptopurine
  • methotrexate platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP- 16); ifosfamide; mitomycin C; n ⁇ toxantrone; vincristine; vinorelbine; navelbine; novantrone; leniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-I l; lopoisomerase inhibitor RFS 2000; difluoromcthylornithinc (DMFO); rctinoic acid; esperamicins; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above.
  • DMFO difluoromcthylornithinc
  • anti-hormonal agents that act to regulate or inhibit hormone action on tumors
  • anti-estrogens including for example tamoxifen, raloxifene, aromatas ⁇ inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxif ⁇ ne, keoxifene, LYl 17018, onapristone, and toreraifene (Faresto ⁇ ); and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goser ⁇ lin; and pharmaceutically acceptable salts, acids or derivatives of any of the above.
  • the cargo moiety delivered in association with the aplamer included in an inventive system may be any of various therapeutic and diagnostic agents which are desired to be delivered to a target.
  • Therapeutic agents which can be included as cargo moieties in the delivery system of the present invention illustratively include but are not limited to therapeutic compounds such as an analgesic, an anesthetic, an antibiotic, an anticonvulsant, an antidepressant, an antimicrobial, an anti -inflammatory, anti-migraine, an antineoplastic, an antiparasitic, an antitumor agent, an antiviral, an anxiolytic, a cytostatic, cytokine, a hypnotic, a metastasis inhibitor, a sedative and a tranquilizer.
  • therapeutic compounds such as an analgesic, an anesthetic, an antibiotic, an anticonvulsant, an antidepressant, an antimicrobial, an anti -inflammatory, anti-migraine, an antineoplastic, an antiparasitic, an anti
  • the aptamers are labeled with a detectable agent, which are administered to a patient for the in vivo imaging of a tumor.
  • a detectable agent which are administered to a patient for the in vivo imaging of a tumor.
  • the specific deliver ⁇ ' of the detectable agent provides a vastly superior means of specific detection of a tumor or desired target cell and decreases any background noise, allowing for the early detection and diagnosis of, for example, a tumor.
  • Diagnostic agents that may be included in the delivery system of the present invention as cargo moieties illustratively include but arc not limited to a contrast agent, a labeled imaging agent such as a radiolabeled imaging agent, and an anti turn oral agent. Combinations of therapeutic compounds may be included, combinations of diagnostic agents may be included, and combinations of both therapeutic and diagnostic agents may be included, Further suitable therapeutic and diagnostic compounds that may be delivered by a system according to the invention may be found in standard pharmaceutical references such as A, R, Gennaro, Remington: The Science and Practice of Pharmacy, Lippincotl Williams & Wilkins, 20th ed. (2003); L. V. Allen, Jr. et al., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, 8th Ed. (Philadelphia, PA: Lippincott, Williams & Wilkins. 2004); J. 0. Hardman et al,,
  • the detectable agent which is associated with the aptamer can also be a therapeutic agent.
  • a radioactive material which can be utilized as an imaging agent, and at the same time is a therapeutic agent when it is delivered locally to the tumor by the aptamer specific for that cell.
  • the aptamer is conjugated to a diagnostic agent and a therapeutic moiety.
  • aptamer targeted si RN A/gene silencing to modulate antitumor immunity by inducing or enhancing antigenicity of target cell:
  • the use of aptamer-oligonucleotides to manipulate tumor immunity is directed to increase the expression of existing antigens or induce expression of novel antigens which would be recognized by the immune system as foreign.
  • aptamcrs to target gene silencing to the appropriate cells in vivo provides a drug/reagent that can be chemically synthesized in cell-free systems which significantly enhances the clinical applicability of this targeting approach (compared to antibody- based targeting), drastically reducing the amount of s ⁇ RN A reagent needed for treatment and consequently the cost-effectiveness and toxicity of the treatment. Furthermore, a key advantage of immune modulating drugs., whether targeted or riot, is that only a fraction of the target cells need to be accessed in vivo for the approach to be successful.
  • RNAi's of the invention can also be obtained using a number of techniques known to those of skill in the art.
  • the siRNA can be chemically synthesized or
  • RNAi's of the invention are chemically synthesized using appropriately protected ribonucleotide phosphoramidites and a conventional DNA/RNA synthesizer.
  • the RNAi can be synthesized as two separate, complementary RNA molecules, or as a single RNA molecule with two complementary regions.
  • Commercial suppliers of synthetic RNA molecules or synthesis reagents include Proligo (Hamburg, Germany), Dharmacon Research (Lafayette, Colo., USA), Pierce Chemical (part of Perbio Science, liockford, HL, USA), Glen Research (Sterling, Va., USA), ChemGenes (Ashland, Mass., USA) and Cruachcm (Glasgow, UK).
  • RNAi can also be expressed from recombinant circular or linear DNA plasmids using any suitable promoter.
  • suitable promoters for expressing RNAi of the invention from a plasmid include, for example, the U ⁇ or Hl RNA pol III promoter sequences and the cytomegalovirus promoter. Selection of other suitable promoters is within the skill in the art.
  • the recombinant plasmids of the invention can also comprise inducible or regulatable promoters for expression of the RNAi in a particular tissue or in a particular intracellular environment.
  • RNAi's of the invention can be expressed from a recombinant plasmid either as two separate, complementary RNA molecules, or as a single RNA molecule with two complementary regions.
  • plasmids suitable for expressing RNAi of the invention are within the skill in the art. See, for example Tuschl, T. (2002), Nat. Biotechnol, 20: 446-448; Brummelkamp T R et al (2002), Science 296: 550-553; Miyagishi M et al. (2002), Nat. Biotechnol. 20: 497-500; Paddison P J et al (2002), Genes Dev. 16:948-958: Lee N S et al, (2002), Nat. Biotechnol. 20: 500-505; and Paul C P et al. (2002), Nat. Biotechnol. 20: 505-508, the entire disclosures of which are herein incorporated by reference.
  • in operable connection with a poiyT termination sequence means that the nucleic acid sequences encoding the sense or anti sense strands are immediately adjacent to the poly T termination signal in the 5' direction. During transcription of the sense or antisense sequences from the plasmid, the poly T termination signals act to terminate transcription. [0116] As used herein, "under the control" of a promoter means that the nucleic acid sequences encoding the sense or antisense strands are located 3' of the promoter, so that the promoter can initiate transcription of the sense or antisense coding sequences.
  • Any viral vector capable of accepting the coding sequences for the siRNA molccule(s) to be expressed can be used, for example vectors derived from adenovirus (AV); adeno-assoeiated virus (AAV); retroviruses (e.g., lcntiviruscs (LV), Rhabdoviruses. murine leukemia vims);
  • AV adenovirus
  • AAV adeno-assoeiated virus
  • retroviruses e.g., lcntiviruscs (LV), Rhabdoviruses. murine leukemia vims
  • the tropism of the viral vectors can also be modified by pseudoiyping the vectors with envelope proteins or other surface antigens from other viruses.
  • an AAV vector of the invention can be pscudo typed with surface proteins from vesicular stomatitis virus (VSV), rabies, Ebola, Mokola, and the like.
  • a suitable AV vector for expressing the RNAi's of the invention a method for constructing the recombinant AV vector, and a method for delivering the vector into target cells, are described in Xia H ei al. (2002), Nat. Biotech. 20: 1006-1010.
  • Suitable AAV vectors for expressing the RNAi's of the invention, methods for constructing the recombinant AAV vector, and methods for delivering the vectors into target cells are described in Samulski R ei al. (1987), J. Virol, 61 : 3096-3101 : Fisher K J er «/. (1996), J. Virol., 70: 520-532: Samulski R el al. (1989), J.
  • RNAi containing a given target sequence can be evaluated using standard techniques for measuring the levels of RNA or protein in cells.
  • RNA of the invention can be delivered to cultured cells, and the levels of target niRNA can be measured by Northern blot or dot blotting techniques, or by quantitative RT-PCR.
  • RNAi-mediated degradation of target mRNA by an siRNA containing a given target sequence can also be evaluated with animal models, such as mouse models.
  • RNAi-medialed degradation of the target mRNA can be detected by measuring levels of the target mliNA or protein in the cells of a subject, using standard techniques for isolating and quantifying mRNA or protein as described above.
  • siRNA molecules target overlapping regions of a desired sense/antisense locus, thereby modulating both the sense and antisens ⁇ transcripts.
  • a composition comprises siRNA molecules, of either one or more, and/or, combinations of siRNAs, siRNAs that overlap a desired target locus, and/or target both sense and antisense (overlapping or otherwise). These molecules can be directed to any target that is desired for potential therapy of any disease or abnormality, Theoretically there is no limit as to which molecule is to be targeted. Furthermore, the technologies taught herein allow for tailoring therapies to each individual.
  • the oligonucleotides can be tailored to individual therapy, for example, these oligonucleotides can be sequence specific for allelic variants in individuals, the up-rcgulation or inhibition of a target can be manipulated in varying degrees, such as for example, 10%, 20%, 40%, 100% expression relative to the control. That is, in some patients it may be effective to increase or decrease target gene expression by 10% versus 80% in another patient.
  • J lip-regulation or inhibition of gene expression may be quantified by measuring either the endogenous target RNA or the protein produced by translation of the target RNA. Techniques for quantifying IiNA and proteins are well known to one of ordinary skill in the art.
  • gene expression is inhibited by at least 10%, preferably by at least 33%, more preferably by at least 50%, and yet more preferably by at least 80%.
  • gene expression is inhibited by at least 90%, more preferably by at least 95%, or by at least 99% up to 100% within cells in the organism, In certain preferred embodiments, gene expression is up-regulated by at least 10%, preferably by at least 33%, more preferably by at least 50%, and yet more preferably by at least 80%, In particularly preferred embodiments, of the invention gene expression is up-regulated by at least 90%, more preferably by at least 95%, or by at least 99% up to 100% within cells in the organism, [0125] Selection of appropriate RNAi is facilitated by using computer programs that automatically align nucleic acid sequences and indicate regions of identity or homology.
  • Such programs are used to compare nucleic acid sequences obtained, for example, by searching databases such as GeiiBank or by sequencing PCR products. Comparison of nucleic acid sequences from a range of species allows the selection of nucleic acid sequences that display an appropriate degree of identity between species.
  • Southern blots are performed to allow a determination of the degree of identity between genes in target species and other species. By performing Southern blots at varying degrees of stringency, as is well known in the art, it is possible to obtain an approximate measure of identity.
  • RNAi that exhibit a high degree of complementarity to target nucleic acid sequences in a subject to be controlled and a lower degree of complementarity to corresponding nucleic acid sequences in other species.
  • One skilled in the art will realize that there is considerable latitude in selecting appropriate regions of genes for use in the present invention.
  • small interfering RNA either as RNA itself or as DNA, is delivered to a cell using aptamers.
  • Figure 2 provides a schematic illustration of aptamcr targeted si RN As.
  • the siRN A or oligonucleotide can be attached to one or more aptamers or encoded as a single molecule so that the 5' to 3' would encode for an aptamer, the siRN A and an aptamer. These can also be attached via linker molecules.
  • the composition can also comprise in a 5'to 3' direction an aptamer attached to another aptamer via a linker which are then attached to the siRNA. These molecules can also be encoded in the same combination.
  • Compositions can include various permutations and combinations.
  • the composition can include siRNAs specific for different polynucleotide targets.
  • the nucleic acid molecules of the present disclosure can be synthesized separately and joined together post-synthetically, for example, by ligation (Moore et ah, Science 256:9923, 1992; Draper et ah, PCT Publication No. WO 93/23569; Shabarova et ah, Nucleic Acids Res, /9:4247, 1991; Bellon et a!.. Nucleosides & Nucleotides 16:951 , 1997; Bellon et al., Bioconjiigaie Chem. #:204, 199 " ), or by hybridization following synthesis or dcprotection.
  • RNAi's can be made as single or multiple transcription products expressed by a polynucleotide vector encoding one or more siRNAs and directing their expression within host cells.
  • An RNAi or analog thereof of this disclosure may be further comprised of a nucleotide, non-nucleotidc, or mixed nucleotidc/non-nuclcotide linker that joins the aptamers and RNAi's.
  • a nucleotide linker can be a linker of more than about 2 nucleotides length up to about 50 nucleotides in length.
  • the nucleotide linker can be a nucleic acid aptamer.
  • a non-nucleotide linker may be comprised of an abasic nucleotide, polyether, polyamine, polyamidc, peptide, carbohydrate, lipid, polyhydrocarbon, or other polymeric compounds (e.g., polyethylene glycols such as those having between 2 and 100 ethylene glycol units). Specific examples include those described by Seela and Kaiser, Nucleic Acids Res. 15:6353, 1990, and Nucleic Acids Res. /5:31 13. 1987; Cload and Schepartz, /. Am. Chera. Soc. /75:6324, 1991; Richardson and Schepartz, J. Am. Chem. Soc.
  • the invention may be used against protein coding gene products as well as non-protein coding gene products.
  • non-protein coding gene products include gene products that encode ribosomal RNAs, transfer RNAs, small nuclear RNAs, small cytoplasmic RNAs, telomerase RNA, RNA molecules involved in DNA replication, chromosomal rearrangement and the like.
  • siRNA oligonucleotide therapies comprise
  • siRNA oligonucleotide which contacts (interacts with) the targeted mRNA from the gene, whereby expression of the gene is modulated.
  • modulation of expression suitably can be a difference of at least about 10% or 20% relative to a control, more preferably at least about 30%, 40%, 50%, 60%, 70%, 80%, or 90% difference in expression relative to a control. It will be particularly preferred where interaction or contact with an siRNA oligonucleotide results in complete or essentially complete modulation of expression relative to a control, e.g., at least about a 95%, 97%, 98%, 99% or 100% inhibition of or increase in expression relative to control.
  • a control sample for determination of such modulation can be comparable cells (in vitro or in vivo) that have not been contacted with the si UNA oligonucleotide.
  • the nucicobases in the siRNA may be modified to provided higher specificity and affinity for a target raRNA.
  • nucleobases may be substituted with LNA monomers, which can be in contiguous stretches or in different positions.
  • the modified siRNA preferably has a higher association constant (K a ) for the target sequences than the complementary sequence. Binding of the modified or non-modified siRNA's to target sequences can be determined in vitro under a variety of stringency conditions using hybridization assays and as described in the examples which follow.
  • a fundamental property of oligonucleotides that underlies many of their potential therapeutic applications is their ability to recognize and hybridize specifically to complementary single stranded nucleic acids employing either Watson-Crick hydrogen bonding (A-T and G-C) or other hydrogen bonding schemes such as the Ho ⁇ gsteen/rcvcrse Hoogsteen mode.
  • Affinity and specificity are properties commonly employed to characterize hybridization characteristics of a particular oligonucleotide. Affinity is a measure of the binding strength of the
  • thermostability T m
  • affinity increases with increasing size (No, of nucleobases) of the oligonucleotide.
  • Specificity is a measure of the ability of the oligonucleotide to discriminate between a fully complementary and a mismatched target sequence. In other words, specificity is a measure of the loss of affinity associated with mismatched nucleobase pairs in the target.
  • siRNA oligonucleotide for modulation (including inhibition) of an mRNA can be readily determined by simple testing.
  • an in vitro or in vivo expression system comprising the targeted mRNA, mutations or fragments thereof, can be contacted with a particular siRNA oligonucleotide (modified or un modified) and levels of expression are compared to a control, that is, using the identical expression system which was not contacted with the siRNA oligonucleotide.
  • oligonucleotides may be used in combinations. For instance, a cocktail of several different siRNA modified and/or unmodified oligonucleotides, directed against different regions of the same gene, may be administered simultaneously or separately.
  • target gene products may be single-stranded or double-stranded DNA or RNA
  • Short dsRNA can be used to block transcription if they are of the same sequence as the start site for transcription of a particular gene. See, for example, Janowski et al. Nature Chemical 2005, 10:1038. Il is understood thai the targcl to which the siRN A oligonucleotides of the invention are directed include allelic forms of the targeted gene and the corresponding mRNAs including splice variants.
  • Preferred mRNA targets include the 5' cap site, tRNA primer binding site, the initiation codon site, the mRNA donor splice site, and the mRNA acceptor splice site,
  • target polynucleotide comprises an mRNA transcript
  • complementary oligonucleotides can hybridize to any desired portion of the transcript.
  • Such oligonucleotides are, in principle, effective for inhibiting translation, and capable of inducing the effects described herein. It is hypothesized that translation is most effectively inhibited by the mRNA at a site at or near the initiation codon.
  • oligonucleotides complementary to the 5'- region of mRNA transcript are preferred.
  • Oligonucleotides complementary to the mRNA, including the initiation codon (the first codon at the 5' end of the translated portion of the transcript), or codons adjacent to the initiation codon are preferred.
  • mRNA includes not only the coding region which carries the information to encode a protein using the three letter genetic code, including the translation start and stop codons, but also associated ribonucleotides which form a region known to such persons as the 5 '-untranslated region, the S'-untranslated region, the 5' cap region, intron regions and intron/exon or splice junction ribonucleotides.
  • oligonucleotides may be formulated in accordance with this invention which are targeted wholly or in part to these associated ribonucleotides as well as to the coding ribonucleotides.
  • the oligonucleotide is targeted to a translation initiation site (AUG codon) or sequences in the coding
  • RNA to be interfered with include all vital functions such as translocation of the RNA to the site for protein translation, actual translation of protein from the RNA, splicing or maturation of the RNA and possibly even independent catalytic activity which may be engaged in by the RNA.
  • the overall effect of such interference with the RNA function is to cause interference with protein expression.
  • Certain preferred oligonucleotides and ap tamers of this invention arc chimeric.
  • oligonucleotides which contain two or more chemically distinct regions, each made up of at least one nucleotide. These oligonucleotides typically contain at least one region of modified nucleotides that confers one or more beneficial properties (such as, for example, increased nuclease resistance, increased uptake into cells, increased binding affinity for the RNA target) and a region that is a substrate for enzymes capable of cleaving RNA:DNA or RNArRNA hybrids.
  • RNase LI is a cellular cndonuclease which cleaves the RNA strand of an RNArDNA duplex.
  • a chimeric oligonucleotide comprises at least one region modified to increase target binding affinity, and, usually, a region that acts as a substrate for RNAse H, Affinity of an
  • oligonucleotide for its target is routinely determined by measuring the T 1n of an oligonucleotide/larget pair, which is the temperature at which the oligonucleotide and target dissociate: dissociation is detected spcctrophotometrically.
  • T 1n the temperature at which the oligonucleotide and target dissociate: dissociation is detected spcctrophotometrically.
  • T 1n the higher the T 1n , the greater the affinity of the oligonucleotide for the target.
  • the region of the oligonucleotide which is modified comprises at least one nucleotide modified at the 2' position of the sugar, preferably a 2'-O-alkyl, 2'-O-alkyl-O-alkyl or 2'-tluoro-modified nucleotide.
  • RNA modifications include 2'-fluoro, 2'-amino and 2' O-mcthyl modifications on the ribose of pyrymidines, abasic residues or an inverted base at the 3' end of the RNA.
  • RNAse H is a cellular endonuclcasc that cleaves the RNA strand of RN A: DNA duplexes; activation of this enzyme therefore results in cleavage of the RNA target, and thus can greatly enhance the efficiency of RNAi inhibition. Cleavage of the RNA target can be routinely demonstrated by gel electrophoresis.
  • the chimeric oligonucleotide is also modified to enhance nuclease resistance.
  • Cells contain a variety of exo- and endo-nucleases which can degrade nucleic acids. A number of nucleotide and nucleoside modifications have been shown to make the oligonucleotide into which they arc incorporated more resistant to nuclease digestion than the native oligodeoxynuclcotide.
  • Nuclease resistance is routinely measured by incubating oligonucleotides with cellular extracts or isolated nuclease solutions and measuring the extent of intact oligonucleotide remaining over time, usually by gel electrophoresis, Oligonucleotides which have been modified to enhance their nuclease resistance survive intact for a longer time than unmodified
  • oligonucleotides A variety of oligonucleotide modifications have been demonstrated to enhance or confer nuclease resistance. Oligonucleotides which contain at least one phosphorothioate modification are presently more preferred. In some cases, oligonucleotide modifications which enhance target binding affinity are also, independently, able to enhance nuclease resistance. Some desirable modifications can be found in De Mesmaeker ei al. Ace. Chem. Res. 1995, 28:366-374.
  • oligonucleotides envisioned for this invention include those comprising modified backbones, for example, phosphorothioates, phosphotriesters. methyl phosphonates, short chain alkyl or cycloalkyl intersugar linkages or short chain hcteroatomic or heterocyclic intersugar linkages.
  • oligonucleotides with phosphorothioale backbones and those with hcleroatom backbones particularly CTl 2 — NH- Q-- CH 2 , CH, ⁇ N(CH 3 )-O ⁇ CH 2 [known as a mcthylene(mcthylimmo) or MMl backbone], CH 2 --O- -N (CHO-CH 2 , CH;. -N (CHj)-N (CHO-CH 2 and O-N (CHj)-CH 2 -CH 2 backbones, wherein the native phosphodiest ⁇ r backbone is represented as O— P— O--CH,).
  • oligonucleotides having morpholino backbone structures are also preferred.
  • oligonucleotides having morpholino backbone structures are also preferred.
  • oligonucleotides having morpholino backbone structures are also preferred.
  • the phosphodiester backbone of the oligonucleotide is replaced with a polyatnide backbone, the nucleobases being bound directly or indirectly to the aza nitrogen atoms of the polyamide backbone (Nielsen et a!. Science 1991, 254, 1497)
  • Oligonucleotides may also comprise one or more substituted sugar moieties.
  • Preferred oligonucleotides comprise one of the following at the T position: OH, SH, SCH,, F, OCN, OCH 3 OCH 3 .
  • hcterocycloalkaryi aminoalkylamino: polyalkylamino; substituted silyl; an RNA cleaving group; a reporter group; an intercalate! a group for improving the pharmacokinetic properties of an oligonucleotide; or a group for improving the pharmacodynamic properties of an oligonucleotide and other substituents having similar properties.
  • a preferred modification includes 2'- methoxyethoxy [2'-0-CH 2 CFl 2 OCH 3 , also known as 2'-O-(2-melhoxy ethyl)] (Martin et a!.. HeIv. CMm.
  • Oligonucleotides may also include, additionally or alternatively, nucleobasc (often referred to in the art simply as “base 1" ) modifications or substitutions.
  • base 1 nucleobasc
  • nucleobases include adenine (A), guanine (G), thymine (T), cytosine (C) and uracil (U).
  • Modified nucleobases include nucleobases found only infrequently or transiently in natural nucleic acids, e.g., hypoxanthine, 6-tnethyladenine, 5 -Me pyritnidines, particularly 5-methylcytosinc (also referred to as 5-methyl-2' dcoxycytosine and often referred to in the art as 5-Me-C), 5-hydroxymethylcylosine (FlMC), giycosyl FlMC and gentobiosyl HMC, as well as synthetic nucleobases, e.g., 2-aminoadenine, 2-(methylamino)adenine, 2- (imidazolylalkyl)adenine, 2 ⁇ (ammoalklyamino)adenme or other heterosubstiluled
  • Another modification of die oligonucleotides of the invention involves chemically linking to the oligonucleotide one or more moieties or conjugates which enhance the activity or cellular uptake of the oligonucleotide.
  • moieties include but are not limited to lipid moieties such as a cholesterol moiety, a cholesteryl moiety (Letsinger et al., Proc. Natl. Acad. ScL USA 1989, 86, 6553), cholic acid (Manoharan ei al. Bioorg. Med. Ch em. Lei.
  • a thioethcr e.g., hexyl-S-tritylthioi (Manoharan et a Ann. N. Y. Acad. Set. 1992, 660, 306; Manoharan et a!. Bioorg. Med. Chem. Let. 1993, 3, 2765), a thiocholcstcrol (Obcrhauscr et al., NucL Acids Res, 1992, 20, 533), an aliphatic chain, e.g.. dodecandiol or undecyl residues (Saison-Behraoaras et al EMBO J. 1991 , 10, 111 ; Kabanov et al. FEBS Lett. 1990. 259, 327; Svinarchuk et al.
  • a phospholipid e.g., di-hexadccyl-rac-glyccrol or triethylammonium l,2-di-O-hexadecyl-rac-glycero-3-H- ⁇ hosphonate ⁇ Manoharan et al. Tetrahedron Lent. 1995, 36, 3651: Shea et al. NucL Acids Res. 1990. 18, 3777), a polyaminc or a polyethylene glycol chain (Manoharan et al. Nucleosides & Nucleotides 1995, 14, 969), or adamantane acetic acid
  • Oligonucleotides comprising lipophilic moieties, and methods for preparing such oligonucleotides are known in the art, for example, U.S. Pat. Nos. 5, 138,045, 5,218, 105 and 5,459,255.
  • oligonucleotides which are chimeric oligonucleotides as hereinbefore defined.
  • the nucleic acid molecule of the present invention is conjugated with another moiety including but not limited to abasic nucleotides, polyether, polyamine, polyamidcs, peptides, carbohydrates, lipid, or polyhydrocarbon compounds. Those skilled in the art will recognize that these molecules can be linked to one or more of any nucleotides comprising the nucleic acid molecule at several positions on the sugar, base or phosphate group.
  • the oligonucleotides used in accordance with this invention may be conveniently and routinely made through the well-known technique of solid phase synthesis. Equipment for such synthesis is sold by several vendors including Applied Biosystems.
  • oligonucleotides Any other means for such synthesis may also be employed; the actual synthesis of the oligonucleotides is well within the talents of one of ordinary skill in the art. It is also well known to use similar techniques to prepare other oligonucleotides such as the phosphorothioatcs and alkylated derivatives. It is also well known Io use similar techniques and commercially available modified araidites and eontrolled-pore glass (CPG) products such as biotin, fluorescein, acridine or psoralen-modified amidiles arid/or CPG (available from Glen Research, Sterling VA) to synthesize fluorescently labeled, biotinylated or other modified oligonucleotides such as cholesterol-modified
  • CPG modified araidites and eontrolled-pore glass
  • oligonucleotides comprised of current chemistries such as MOE, ANA, FANA, PS etc (Recent advances in the medical chemistry of antisense oligonucleotide by Uhlman, Current Opinions in Drug Discovery & Development 2000 VoI 3 No 2). This can be achieved by substituting some of the monomers in the current oligonucleotides by LNA monomers.
  • the LNA modified oligonucleotide may have a size similar to the parent compound or may be larger or preferably smaller.
  • LNA-modii ⁇ ed oligonucleotides contain less than about 70%, more preferably less than about 60%, most preferably less than about 50%) LNA monomers and that their sizes are between about 10 and 25 nucleotides, more preferably between about 12 and 20 nucleotides,
  • siRNA's target genes that prevent the normal expression or, if desired, over expression of genes thai are of therapeutic interest as described above.
  • the term "overexpressing" when used in reference to the level of a gene expression is intended to mean an increased accumulation of the gene product in the overexpressing cells compared to their levels in counterpart normal cells. Overexpression can be achieved by natural biological phenomenon as well as by specific modifications as is the case with genetically engineered cells. Overexpression also includes the achievement of an increase in cell survival polypeptide by either endogenous or exogenous mechanisms.
  • Overexpression by natural phenomenon can result by, for example, a mutation which increases expression, processing, transport, translation or stability of the RNA as well as mutations which result in increased stability or decreased degradation of die polypeptide. Such examples of increased expression levels are also examples of endogenous mechanisms of overexpression.
  • a specific example of a natural biologic phenomenon which results in overexpression by exogenous mechanisms is the adjacent integration of a retrovirus or transposon.
  • Overexpression by specific modification can be achieved by, for example, the use of siRNA oligonucleotides described herein.
  • siRNA polynucleotide may be constructed in a number of different ways provided that it is capable of interfering with the expression of a target protein.
  • the siRNA may be constructed in a number of different ways provided that it is capable of interfering with the expression of a target protein.
  • polynucleotide generally will be substantially identical (although in a complementary
  • the minimal identity will typically be greater than about 80%, greater than about 90%, greater than about 95% or about 100% identical.
  • the aptamer-oligonuclcotides in some embodiment, further comprise non- nucleic acid moieties/moieties which may take any number of diverse forms depending on the function desired. For example, modifications introduced in the oligonucleotide backbone of the aptamer-siRNA chimeras: (i) To promote cytoplasmic delivery of the endocytos ⁇ d aplamer- siRNAs, the aptamer-siRNA ODNs are conjugated to peptides which promote cytoplasmic translocation from endosomes, such the HIV derived tat peptide, a fusogenic peptide from influenza hemagglutinin protein, a 9mer Arg oligopeptide and others, (ii) To increase
  • bioavailability the aptamer-siRNA chimeras are conjugated to cholesterol or polyethylene glycol.
  • the moieties include natural polymers, synthetic polymers, natural ligands and synthetic ligands, as well as combinations of any and all of the foregoing.
  • suitable members may be modified or unmodified.
  • Natural polymers can be selected from a polypeptide, a protein, a polysaccharide, a fatty acid, and a fatty acid ester as well as any and all combinations of the foregoing.
  • homopolymers and heteropolymers may be employed. Such homopolymers and heteropolymers are in many ways preferred when they carry a net negative charge or a net positive charge.
  • the above-described construct of the present invention can be designed to exhibit a further and additional biological activity which is usefully imparted by incorporating at least one or more modified nucleotides, nucleotide analogs, nucleic acid moieties, ligands or a combination of any or all of these.
  • Such biological activity may itself take a number of forms, including nuclease resistance, cell recognition, cell binding, and cellular (cytoplasmic) or nuclear localization.
  • Ligands or chemical modifications can be attached Io the nucleic acid, modified nucleic acid or nucleic acid analogue by modification of the sugar, base and phosphate moieties of the constituent nucleotides (Engelhardt ct al,, U.S. Pat. No. 5,260,433, fully incorporated herein by reference) or to a non nucleic acid segment of such as polysaccharide, polypeptide and other polymers both natural and synthetic. Modifications of sugar and phosphate moieties can be preferred sites for terminal binding of ligands or chemical modifications and other moieties. Modifications of the base moieties can be utilized for both internal or terminal binding of ligands or chemical modifications and other moieties.
  • Chemical modification can be limited to a specific segment of the construct such as a tail or a gap, or dispersed throughout the molecule,
  • Ligands or chemical modifications being any chemical entity, natural or synthetic, which can be utilized in this invention include macromolecules greater than 20,000 M.W, as well as small molecules less that 20,000 M.W.
  • the Ugarid or ligands can include both raacroniolecules and small molecules.
  • Macromolecules which can be utilized include a variety of natural and synthetic polymers including peptides and proteins, nucleic acids, polysaccharides, lipids, synthetic polymers including poiyanions, polycations, and mixed polymers.
  • Small molecules include oligopeptides, oligonucleotides, monosaccharides, oligosaccharides and synthetic polymers including poiyanions, polycations, lipids and mixed polymers. Small molecules include mononucleotides, oligonucleotides, oligopeptides, oligosaccharides, monosaccharides, lipids, sugars, and other natural and synthetic moieties.
  • Ligands and chemical modifications provide useful properties for nucleic acid transfer such as 1) cell targeting moieties, 2) moieties which facilitate cellular uptake, 3) moieties specifying intracellular localization, 4) moieties which facilitate incorporation into cellular nucleic acid and 5) moieties which impart nuclease resistance,
  • the aptanier-oligonucleotide molecules comprise one or more moieties comprising one or more of: polylysine, polyarginine, Antennapedia-derived peptides, HIV derived tat peptide, a iusogenic peptide from influenza hemagglutinin protein, a 9mcr Arg oligopeptide, peptide transporters, peptide transduction domains, intracellular localization domain sequences, or combinations thereof.
  • Moieties which facilitate cellular uptake include inactivated viruses such as adenovirus (Cristiano et al., 1993, Proc Nm'l Acad Sci USA 90:2122: Curiel et ai, 1991, P roc N at' I Acad Sci USA 88:8850, ail of which are incorporated by reference); vims components such as the hemaglutinating protein of influenza virus and a peptide fragment from it, the hemagglutinin HA-2 N-terminal fusogcnic peptide (Wagner et al, 1992, Proc Natl Acad ' USA 89:7934, also incorporated herein by reference).
  • Moieties which specify cellular location include: a) nuclear proteins such as histones; b) nucleic acid species such as the snRNAs Ul and U2 which associate with cytoplasmic proteins and localize in the nucleus (Zieve and Sautereauj, 1990, Biochemistry and Molecular Biology 25; 1);4) moieties which facilitate incorporation into cellular nucleic acid include: a) proteins which function in integration of nucleic acid into DNA. These include integrase site specific recombinases (Argos et al., 1986, EMBO Journal 5: 433); and b) homologous nucleotide sequences to cellular DNA to promote site specific integration,
  • Ligands or chemical modifications can be introduced into aptanier-oligonucleotide molecules either a) directly by conjugation, b) by enzymatic incorporation of modified nucleoside triphosphates c) by reaction with reactive groups present in constituent nucleotides and d) by incorporation of modified segments.
  • These processes include both chemical and enzymatic methods. Enzymatic methods include primer extension, RNA and DNA ligation, random priming, nick translation, polymerase chain reaction, RNA labeling methods utilizing T " , T3 and 5P6 polymerases, terminal addition by terminal transferase.
  • Chemical methods include direct attachment of ligands or chemical modifications to activated groups in the nucleic acid such as allylaminc. bromo, thio and amino: incorporation of chemically modified nucleotides during chemical synthesis of nucleic acid, chemical end labeling; labeling of nucleic acid with enzymes.
  • the aptamcr-oligonucleotide construct of the present invention carries a net positive charge or a net negative charge. Further, the construct can be neutral or even hydrophobic. It should not be overlooked that the construct may comprise unmodified nucleotides and at least one other member or element selected from one or more nucleotide analogs and non-nucleic acid moieties, or both.
  • Aptamers are high affinity single-stranded nucleic acid ligands which can be isolated from combinatorial libraries through an iterative process of in vitro selection known as
  • aptamers exhibit specificity and avidity comparable to or exceeding that of antibodies, and can be generated against most targets. Unlike antibodies, aptamers, or in this instance aptamer-oligonuclcotides fusions, can be synthesized in a chemical process and hence offer significant advantages in terms of reduced production cost and much simpler regulatory approval process. Also, aptamers- siRNAs are not expected to exhibit significant immunogenicily in vivo.
  • the siRNA is linked to at least one aptatner which is specific for a desired cell and target molecule.
  • the RNAi's are combined with two aptamers. For example, Figure 2. The various permutations and combinations for combining aptamers and RNAi's is limited only by the imagination of the user,
  • Methods of the present disclosure do not require a prion knowledge of the nucleotide sequence of every possible gene variant (including mRNA splice variants) targeted by the RNAi or analog thereof.
  • Aptamers specific for a given biomolcculc can be identified using techniques known in the art. See, e.g., Toole et a!. (1992) PCT Publication No. WO 92/14843; Tu ⁇ rk and Gold (1991) PCT Publication No. WO 91/19813: Weintraub and Hutchinson (1992) PCT Publication No. 92/05285; and Ellington and Szostak, Nature 346:818 (1990). Briefly, these techniques typically involve the complexalion of the molecular target with a random mixture of oligonucleotides.
  • the aptamcr-molec ⁇ lar target complex is separated from the uncomplcxcd oligonucleotides.
  • the aptatner is recovered from the separated complex and amplified. This cycle is repeated to identify those ap tamer sequences with the highest affinity for the molecular target.
  • the SELEX lM process is a method for the in vitro evolution of nucleic acid molecules with highly specific binding to target molecules and is described in, e.g., U.S. Pat. No. 5,270,163 (sec also WO 91/19813) entitled "Nucleic Acid Ligands".
  • Each SELEX-identified nucleic acid ligand is a specific ligand of a given target compound or molecule.
  • the SELEXTM process is based on the unique insight that nucleic acids have sufficient capacity for forming a variety of two- and three-dimensional structures and sufficient chemical versatility available within their monomers to act as ligands (form specific binding pairs) with virtually any chemical compound, whether monomeric or polymeric. Molecules of any size or composition can serve as targets.
  • oligonucleotides comprising randomized sequences derived from chemical synthesis on a standard DNA synthesizer.
  • the oligonucleotides can be modified or unmodified DNA, RNA or DN A/RN A hybrids.
  • the pool comprises 100% random or partially random oligonucleotides. In other examples, the pool comprises random or partially random
  • the pool comprises random or partially random oligonucleotides containing at least one fixed sequence and/or conserved sequence at its 5' and/or 3' end which may comprise a sequence shared by all the molecules of the oligonucleotide pool.
  • Fixed sequences are sequences common to oligonucleotides in the pool which are incorporated for a pre-selected memepos ⁇ such as, CpG motifs, hybridization sites for PCR primers, promoter sequences for RNA polymerases (e.g., T3, T4, T7, and SP6), restriction sites, or honiopolymeric sequences, such as poly A or poly T tracts, catalytic cores, sites for selective binding to affinity columns, and other sequences to facilitate cloning and/or sequencing of an oligonucleotide of interest.
  • conserveed sequences are sequences, other than the previously described fixed sequences, shared by a number of aptamcrs that bind to the same target.
  • the oligonucleotides of the pool preferably include a randomized sequence portion as well as fixed sequences necessary for efficient amplification.
  • the oligonucleotides of the starting poo! contain fixed 5' and 3' terminal sequences which flank an interna! region of 30- 50 random nucleotides.
  • the randomized nucleotides can be produced in a number of ways including chemical synthesis and size selection from randomly cleaved cellular nucleic acids. Sequence variation in lest nucleic acids can also be introduced or increased by mutagenesis before or during the selection/amplification iterations.
  • the random sequence portion of the oligonucleotide can be of any length and can comprise ribonucleotides and/or deoxyribonucleotidcs and can include modified or non-natural nucleotides or nucleotide analogs. See, e.g., U.S. Pat. No. 5,958,691 ; U.S. Pat. No. 5,660,985; U.S. Pat. No. 5,958,691: U.S. Pat. No. 5,698,687; U.S. Pat. No. 5,817,635; U.S. Pat. No.
  • Random oligonucleotides can be synthesized from phosphodi ester-linked nucleotides using solid phase oligonucleotide synthesis techniques well known in the art. See, e.g., Froehler et a!., Nucl. Acid Res. 14:5399-5467 (1986) and Froehler et al, Tel. LeU. 27:5575-5578 (1986). Random oligonucleotides can also be synthesized using solution phase methods such as triester synthesis methods. See, e.g., Sood et ciL, Nucl Acid Res.
  • the starting library of oligonucleotides may be generated by automated chemical synthesis on a DNA synthesizer. To synthesize randomized sequences, mixtures of all four nucleotides are added at each nucleotide addition step during the synthesis process, allowing for random incorporation of nucleotides. As stated above, in one embodiment, random
  • oligonucleotides comprise entirely random sequences; however, in other embodiments, random oligonucleotides can comprise stretches of nonrandom or partially random sequences. Partially random sequences can be created by adding the four nucleotides in different molar ratios at each addition step.
  • the starting library of oligonucleotides may be either RNA or DNA, In those instances where an RNA library is to be used as the starting library it is typically generated by transcribing a DNA library in vitro using T7 RNA polymerase or modified T7 RNA polymerases and purii ⁇ ed.
  • the SELEX fM method includes steps of: (a) contacting the mixture with the target under conditions favorable for binding; (b) partitioning unbound nucleic acids from those nucleic acids which have bound specifically to target molecules; (c) dissociating the nucleic acid-target complexes; (d) amplifying the nucleic acids dissociated from the nucleic acid-target complexes to yield a iigand-enriched mixture of nucleic acids; and (e) reiterating the steps of binding, partitioning, dissociating and amplifying through as many cycles as desired to yield highly- specific, high affinity nucleic acid ligands to the target molecule.
  • the SELEX LVl method further comprises the steps of: (i) reverse transcribing the nucleic acids dissociated from the nucleic acid-target complexes before amplification in step (d); and (ii) transcribing the amplified nucleic acids from step (d) before restarting the process,
  • a nucleic acid mixture comprising, for example, a 20 nucleotide randomized segment can have 4 z0 candidate possibilities. Those which have the higher affinity constants for the target are most likely to bind to the target.
  • a second nucleic acid mixture is generated, enriched for the higher binding affinity candidates. Additional rounds of selection progressively favor the best ligands until the resulting nucleic acid mixture is predominantly composed of only one or a few sequences. These can then be cloned, sequenced and individually tested for binding affinity as pure ligands or aptamers.
  • Cycles of selection and amplification arc repeated until a desired goal is achieved. In the most general case, selection/amplification is continued until no significant improvement in binding strength is achieved on repetition of the cycle.
  • the method is typically used to sample approximately K) 14 different nucleic acid species but may be used to sample as many as about 1(V 8 different nucleic acid species, Generally, nucleic acid aptam ⁇ r molecules are selected in a 5 to 20 cycle procedure. In one embodiment, heterogeneity is introduced only in the initial selection stages and does not occur throughout the replicating process.
  • the selection process is so efficient at isolating those nucleic acid ligands that bind most strongly to the selected target, that only one cycle of selection and amplification is required.
  • Such an efficient selection may occur, for example, in a chromatographic -type process wherein the ability of nucleic acids to associate with targets bound on a column operates in such a manner that the column is sufficiently able to allow separation and isolation of the highest affinity nucleic acid ligands.
  • the target-specific nucleic acid ligand solution may include a family of nucleic acid structures or motifs that have a number of conserved sequences and a number of sequences which can be substituted or added without significantly affecting the affinity of the nucleic acid ligands to the target.
  • nucleic acid primary, secondary and tertiary structures are known to exist.
  • the structures or motifs that have been shown most commonly to be involved in non- Watson- Crick type interactions are referred to as hairpin loops, symmetric and asymmetric bulges, pseudoknots and myriad combinations of the same. Almost all known cases of such motifs suggest that they can be formed in a nucleic acid sequence of no more than 30 nucleotides.
  • SELEX j M procedures with contiguous randomized segments be initiated with nucleic acid sequences containing a randomized segment of between about 20 to about 50 nucleotides and in some embodiments, about 30 to about 40 nucleotides,
  • the 5'-fixed:random:3'-fixed sequence comprises a random sequence of about 30 to about 50 nucleotides
  • the core SELEX IlVl method can be modified to achieve a number of specific objectives.
  • U.S. Pat, No. 5,707,796 describes the use of SELEX ljVl in conjunction with gel electrophoresis to select nucleic acid molecules with specific structural characteristics, such as bent DNA.
  • U.S. Pat. No. 5,763, 177 describes SELEX 1M based methods for selecting nucleic acid ligands containing photo reactive groups capable of binding and/or photo-cross linking to and/or photo-inactivating a target molecule.
  • Count ⁇ r-SELEX JM is a method for improving the specificity of nucleic acid ligands to a target molecule by eliminating nucleic acid ligand sequences with cross-reactivity to one or more non-target molecules.
  • Counler-SELEX IM is comprised of the steps of: (a) preparing a candidate mixture of nucleic acids; (b) contacting the candidate mixture with the target, wherein nucleic acids having an increased affinity to the target relative to the candidate mixture may be partitioned from the remainder of the candidate mixture; (c) partitioning the increased affinity nucleic acids from the remainder of the candidate mixture: (d) dissociating the increased affinity nucleic acids from the target; ( ⁇ ) contacting the increased affinity nucleic acids with one or more non-target molecules such that nucleic acid ligands with specific affinity for the non-target molecuie(s) are removed; and (f) amplifying the nucleic acids with specific affinity only to the target molecule to yield a mixture of nucleic acids
  • oligonucleotides in their phosphodiester form may be quickly degraded in body fluids by intracellular and extracellular enzymes such as endonucl eases and exonuclease before the desired effect is manifest.
  • the SELEXTM method thus encompasses the identification of high-affinity nucleic acid ligands containing modified nucleotides conferring improved characteristics on the ligand, such as improved hi vivo stability or improved delivery
  • modifications include chemical substitutions at the ribose and/or phosphate and/or base positions.
  • one or more modifications of the nucleic acid ligands contemplated in this invention include, but are not limited to, those which provide other chemical groups that incorporate additional charge, polarizability, hydrophobicity, hydrogen bonding, electrostatic interaction, and fluxionality to the nucleic acid ligand bases or to the nucleic acid ligand as a whole.
  • Modifications to generate oligonucleotide populations which are resistant to nucleases can also include one or more substitute internuclcotidc linkages, altered sugars, altered bases, or combinations thereof.
  • Such modifications include, but are not limited to, Imposition sugar modifications, 5-position pyrimidine modifications, 8-position purine modifications, modifications at exocycl ⁇ c amines, substitution of 4-thiouridine, substitution of 5-bromo or 5- iodo-uracil; backbone modifications, phosphorothioate or alkyl phosphate modifications, methyjations, and unusual base-pairing combinations such as the isobases isocylidine and isoguanosine. Modifications can also include 3' and 5' modifications such as capping.
  • oligonucleotides are provided in which the P(O)O group is replaced by P(O)S ("thioate"), P(S)S ("dithi consult"), P(O)NR 2 ("amidate"), P(O)R, P(O)OR', CO or CH 2 ("formacetal") or 3'-amine (— NH--CH2-CH2-), wherein each R or R' is independently H or substituted or unsubstiluted alkyl.
  • Linkage groups can be attached to adjacent nucleotides through an—0—, -N-, or— S— linkage. Not all linkages in the oligonucleotide are required to be identical.
  • the term phosphorothioate encompasses one or more non-bridging oxygen atoms in a phosphodiester bond replaced by one or more sulfur atom.
  • the oligonucleotides comprise modified sugar groups, for example, one or more of the hydroxyl groups is replaced with halogen, aliphatic groups, or functionalized as ethers or amines.
  • the 2'-position of the furanose residue is substituted by any of an O-methyl, Q-alkyl, O-allyl, S-alkyl, S-allyl, or halo group.
  • Pre- SELEX 13'1 process modifications or those made by incorporation into the SELEX rM process yield nucleic acid ligands with both specificity for their SELEXTM target and improved stability, e.g., in vivo stability.
  • Post- SELEX lM process modifications made to nucleic acid ligands may result in improved stability, e.g., in vivo stability without adversely affecting the binding capacity of the nucleic acid ligand.
  • the SELEX lM method encompasses combining selected oligonucleotides with other selected oligonucleotides and non-oligonucleotide functional units as described in U.S. Pat. No. 5,637,459 and U.S. Pat. No. 5,683,867.
  • the SEL.EXTM method further encompasses combining selected nucleic acid ligands with lipophilic or non-immunogenic high molecular weight compounds in a diagnostic or therapeutic complex, as described, e.g., in U.S. Pat. No. 6,011,020, U.S. Pat. No. 6,051,698, and PCT Publication No. WO 98/18480.
  • nucleic acid ligands to small, flexible peptides via the SELEXTM method can also be used in embodiments of the invention.
  • Small peptides have flexible structures and usually exist in solution in an equilibrium of multiple conformers.
  • the aptamcrs with specificity and binding affinity to the target(s) of the present invention are typically selected by the SELEX 1M process as described herein. As part of the SELEX. TM. process, the sequences selected to bind to the target can then optionally be minimized to determine the minimal sequence having the desired binding affinity.
  • the selected sequences and/or the minimized sequences are optionally optimized by performing random or directed mutagenesis of the sequence to increase binding affinity or alternatively to determine which positions in the sequence are essential for binding activity, Additionally, selections can be performed with sequences incorporating modified nucleotides to stabilize the aptamer molecules against degradation in vivo.
  • aptamer- RN Ai compositions enter cells and sub-cellular compartments.
  • further aptamers can be obtained using various methods.
  • a variation of the 8ELEX JjVl process is used to discover aptamers that are able to enter cells or the sub-cellular compartments within cells. These deliver)' aptamers will allow or increase the propensity of an oligonucleotide to enter or be taken up by a cell.
  • the method comprises the ability to selectively amplify aptamers thai have been exposed to the interior of a cell and became modified in some fashion as a result of that exposure. Such modifications include functioning as a template for template-dependent polymerization.
  • SELEX lM permits the discovery of aptamers that are: (i) completely specific with regard to the kind of cell or sub-c ⁇ lluiar compartment, such as the nucleus or cytoplasm, that they permit entry to, (ii) completely generic, or (iii) partially specific,
  • One potential strategy is to substitute cell-association for cell entry, and after incubation of the library with the cells and subsequent washing of the cells, amplify the library members that remain associated with the cells.
  • this may not distinguish between aptamers that permit genuine cell entry and other trivial solutions to the cell-association problem such as binding to the exterior of the cell membrane, entering, but riot leaving, the cell membrane and being taken up by, but not leaving, the endosome.
  • An alternative strategy is to select for some kind of transformation of the oligonucleotide library member that could happen only in the cytoplasm or other sub-cellular compartment, optionally because the library member is conjugated to a transformable entity, and then selectively amplifying the transformed library members.
  • markers include, but are not limited to: reverse transcription, RNaseH, kinase, ⁇ '-phosphorylation, 5'-dephosphorylation, translation-dependent, post-transcriptional modification to give rcstrictable cDNA, transcription- based, ubiquitination, ultraeentrifugation, or utilizing the endogenous protein kinase CIp 1.
  • library members can have a designed hairpin structure at their 3 '-terminus that will reverse-transcribe without a primer.
  • Reverse transcriptase activity is introduced into the cytoplasm using a protein expression vector or vims.
  • the selective amplification of reverse- transcribed sequences is achieved by using a nucleotide composition that will not amplify directly by, for example, PCR such as completely or partially 2'-OH or 2OMe RNA and omitting an RT step from the procedure.
  • the compositions of the invention target desired nucleic acid sequences.
  • Any desired target nucleic acid sequences can be identified by a variety of methods such as SAGE.
  • SAGE is based on several principles.
  • a short nucleotide sequence tag (9 to 10 b.p.) contains sufficient information content to uniquely identify a transcript provided it is isolated from a defined position within the transcript. For example, a sequence as short as 9 b.p. can distinguish 262,144 transcripts given a random nucleotide distribution at the tag site, whereas estimates suggest that the human genome encodes about 80,000 to 200,000 transcripts (Fields, el ah, Nature Genetics, 7:345 1994).
  • the size of the tag can be shorter for lower eukaryotes or prokaryotcs, for example, where the number of transcripts encoded by the genome is lower.
  • a tag as short as 6-7 b.p. may be sufficient for distinguishing transcripts in yeast.
  • serial analysis of the sequence tags requires a means to establish the register and boundaries of each tag.
  • the concept of deriving a defined tag from a sequence in accordance with the present invention is useful in matching tags of samples to a sequence database.
  • a computer method is used to match a sample sequence with known sequences.
  • the tags are used to uniquely identify gene products. This is due to their length, and their specific location (3'j in a gene from which they are drawn.
  • the full length gene products can be identified by matching the tag to a gene data base member, or by using the tag sequences as probes to physically isolate previously unidentified gene products from cDNA libraries.
  • the methods by which gene products are isolated from libraries using DNA probes are well known in
  • Examples of methods of gene expression analysis known in the art include DNA arrays or microarrays (Brazraa and ViIo, FEBS Lett., 2000, 480, 17-24; Celis, et ah, I 7 EBS Lett., 2000, 480, 2-16), READS (restriction enzyme amplification of digested cDNAs) (Prashar and
  • siRNA oligonucleotides that selectively bind to variants of target gene expression products.
  • a "variant" is an alternative form of a gene, Variants may result from at least one mutation in the nucleic acid sequence and may result in altered mRN As or in polypeptides whose structure or function may or may not be altered. Any given natural or recombinant gene may have none, one, or many allelic forms. Common mutational changes that give rise to variants are generally ascribed to natural deletions, additions, or substitutions of nucleotides. Each of these types of changes may occur alone, or in combination with the others, one or more times in a given sequence.
  • Sequence similarity searches can be performed manually or by using several available computer programs known to those skilled in the art.
  • Blast and Smith- Waterman algorithms which are available and known to those skilled in the art, and the like can be used.
  • Blast is NCBI's sequence similarity search tool designed to support analysis of nucleotide and protein sequence databases. Blast can be accessed through the world wide web of the Internet, at, for example, ncbi.nlm.nih.gov/BLAST/.
  • the GCG Package provides a local version of Blast that can be used cither with public domain databases or with any locally available searchable database.
  • GCG Package v9.0 is a commercially available software package that contains over 100 interrelated software programs that enables analysis of sequences by editing, mapping, comparing and aligning them.
  • Other programs included in the GCG Package include, for example, programs which facilitate RNA secondary structure predictions, nucleic acid fragment assembly, and evolutionary analysis, In addition, the most prominent genetic databases
  • GCG GenBank, EMBL, PIR, and SW(SS-PROT) are distributed along with the GCG Package and are fully accessible with the database searching and manipulation programs.
  • GCG can be accessed through the Internet at, for example, http://www.gcg.com/.
  • Fetch is a tool available in GCG that can get annotated GenBank records based on accession numbers and is similar to Entrez.
  • Gen eWorld 2.5 is an automated, flexible, high-throughput application for analysis of polynucleotide and protein sequences. GeneWorld allows for automatic analysis and annotations of sequences. Like GCG, Gen ⁇ W r orld incorporates several tools for homology searching, gene finding, multiple sequence alignment, secondary structure prediction, and motif identification.
  • GeneThesaurus 1.0 JM is a sequence and annotation data subscription service providing information from multiple sources, providing a relational data model for public and local data.
  • BlastParse is a PERL script running on a UNIX platform that automates the strategy described above. BlastParse takes a list of target accession numbers of interest and parses all the GenBank fields into "tab-delimited” text that can then be saved in a "relational database” format for easier search and analysis, which provides flexibility. The end result is a series of completely parsed GenBank records that can be easily sorted, filtered, and queried against, as well as an annotations-relational database,
  • paralogs can be identified for designing the appropriate siKJNA oligonucleotide.
  • Paralogs are genes within a species that occur due to gene duplication, but have evolved new functions, and are also referred to as isotypes.
  • the polynucleotides of this invention can be isolated using the technique described in the experimental section or replicated using PCR.
  • the PCR technology is the subject matter of U.S. Pat. Nos. 4,683,195, 4,800,159, 4,754,065, and 4,683,202 and described in PCR: The
  • this invention also provides a process for obtaining the polynucleotides of this invention by providing the linear sequence of the polynucleotide, nucleotides, appropriate primer molecules, chemicals such as enzymes and instructions for their replication and chemically replicating or linking the nucleotides in the proper orientation to obtain the polynucleotides.
  • these primer molecules such as enzymes and instructions for their replication and chemically replicating or linking the nucleotides in the proper orientation to obtain the polynucleotides.
  • polynucleotides are further isolated. Still further, one of skill in the art can insert the
  • polynucleotide into a suitable replication vector and insert the vector into a suitable host cell (prokaryotic or eukaryotic) for replication and amplification.
  • a suitable host cell prokaryotic or eukaryotic
  • the DNA so amplified can be isolated from the cell by methods well known to those of skill in the art.
  • a process for obtaining polynucleotides by this method is further provided herein as well as the polynucleotides so obtained.
  • Another suitable method for identifying targets for the aptamer-RNAi compositions includes contacting a test sample with a cell expressing a receptor or gene thereof, an allele or fragment thereof; and detecting interaction of the test sample with the gene, an allele or fragment thereof, or expression product of the gene, an allele or fragment thereof.
  • the desired gene, an allele or fragment thereof, or expression product of the gene, an allele or fragment thereof suitably can be detectabiy labeled e.g. with a fluorescent or radioactive component.
  • a cell from a patient is isolated and contacted with a drug molecule that modulates an immune response.
  • the genes, expression products thereof are monitored to identify which genes or expression products are regulated by the drug.
  • Interference RNA 's can then b ⁇ synthesized to regulate the identified genes, expression products that are regulated by the drug and thus, provide therapeutic oligonucleotides.
  • These can be tailored Io individual patients, which is advantageous as different patients do not effectively respond to the same drugs equally, Thus, the oligonucleotides would provide a cheaper and individualized treatment than conventional drag treatments.
  • hybridization with oligonucleotide probes that are capable of detecting polynucleotide sequences, including genomic sequences, encoding desired genes or closely related molecules may be used to identify target nucleic acid sequences.
  • the specificity of the probe whether it is made from a highly specific region, e.g., the 5' regulatory region, or from a less specific region, e.g., a conserved motif, and the stringency of the hybridization or amplification (maximal, high, intermediate, or low), will determine whether the probe identifies only naturally occurring sequences encoding genes, allelic variants, or related sequences.
  • Probes may also be used for the detection of related sequences, and should preferably have at least 50% sequence identity or homology to any of the identified genes encoding sequences, more preferably at least about 60, 70, 75, 80, 85, 90 or 95 percent sequence identity to any of the identified gene encoding sequences (sequence identity determinations discussed above, including use of BLAST program).
  • the hybridization probes of the subject invention may be DNA or RNA and may be derived from the sequences of the invention or from genomic sequences including promoters, enhancers, and in Irons of the gene.
  • homologous refers to the sub unit sequence similarity between two polymeric molecules, e.g., between two nucleic acid molecules such as two DNA molecules, or two polypeptide molecules. When a subunit position in both of the two molecules is occupied by the same monomelic subunit (e.g., if a position in each of two DNA molecules is occupied by adenine) then they arc homologous at that position. The homology between two sequences is a direct function of the number of matching or homologous positions.
  • Means for producing specific hybridization probes for polynucleotides encoding target genes include the cloning of polynucleotide sequences encoding target genes or derivatives into vectors for the production of mRNA probes. Such vectors are known in the art, are
  • RNA polymerases commercially available, and may be used to synthesize RNA probes in vitro by means of the addition of the appropriate RNA polymerases and the appropriate labeled nucleotides.
  • Hybridization probes may be labeled by a variety of reporter groups, for example, by
  • radionuclides such as 32 P or 32 S
  • enzymatic labels such as alkaline phosphatase coupled to the probe via avidin-biotin coupling systems, fluorescent labeling, and the like
  • the polynucleotide sequences encoding a target gene may be used in Southern or Northern analysis, dot blot, or other membrane-based technologies; in PCR technologies; in dipstick, pin, and multiformat ELISA-like assays; and in microarrays utilizing fluids or tissues from patients to detect altered target gene expression.
  • Gel-based mobility-shift analyses may be employed. Other suitable qualitative or quantitative methods are well known in the art.
  • genes, or variants thereof can be verified using techniques well known in the art. Examples include but are not limited to, nucleic acid sequencing of amplified genes, hybridization techniques such as single nucleic acid polymorphism analysis (SNP), microarrays wherein the molecule of interest is immobilized on a biochip. Overlapping cDNA clones can be sequ ⁇ nced by the dideoxy chain reaction using fluorescent dye terminators and an ABl sequencer (Applied Biosystems, Foster City, Calif.). Any type of assay wherein one component is immobilized may be carried out using the substrate platforms of the invention. Bioassays utilizing an immobilized component arc well known in the art.
  • assays utilizing an immobilized component include for example, immunoassays, analysis of protein-protein interactions, analysis of protein-nucleic acid interactions, analysis of nucleic acid-nucleic acid interactions, receptor binding assays, enzyme assays, phosphorylation assays, diagnostic assays for determination of disease state, genetic profiling for drag compatibility analysis, SNP detection, etc.
  • nucleic acid sequence capable of binding to a biotnoiecule of interest can be achieved by immobilizing a library of nucleic acids onto the substrate surface so that each unique nucleic acid was located at a defined position to form an array. The array would then be exposed to the biomolecule under conditions which favored binding of the biomolecule to the nucleic acids. Non-specifically binding biomolecuies could be washed away using mild to stringent buffer conditions depending on the level of specificity of binding desired. The nucleic acid array would then be analyzed to determine which nucleic acid sequences bound to the biomolccule. Preferably the biomolecuies would carry a fluorescent tag for use in detection of the location of the bound nucleic acids.
  • An assay using an immobilized array of nucleic acid sequences may be used for determining the sequence of an unknown nucleic acid: single nucleotide polymorphism (SNP) analysis; analysis of gene expression patterns from a particular species, tissue, cell type, etc.; gene identification; etc.
  • SNP single nucleotide polymorphism
  • oligonucleotides designed from the sequences encoding a desired gene expression product may involve the use of PCR. These oligomers may be chemically synthesized, generated cnzymatically, or produced in vitro. Oligomers will preferably contain a fragment of a polynucleotide encoding the expression products, or a fragment of a polynucleotide complementary to the polynucleotides, and will be employed under optimized conditions for identification of a specific gene. Oligomers may also be employed under less stringent conditions for detection or quantitation of closely-related DNA or RNA sequences.
  • oligonucleotides or longer fragments derived from any of the polynucleotide sequences may be used as targets in a microarray.
  • the microarray can be used to monitor the identity and/or expression level of large numbers of genes and gene transcripts simultaneously to identify genes with which target genes or its product interacts and/or to assess the efficacy of candidate aptam ⁇ r-RNAi compositions in regulating expression products of genes that mediate, for example, tumor specific immune responses. This information may be used to determine gene function, and to develop and monitor the activities of compositions.
  • Microarrays may be prepared, used, and analyzed using methods known in the art (see, e.g., Brennan et ⁇ /., 1995, U.S. Pat. No. 5,474,796; Schena et a!., 1996, Proc. Natl. Acad ScL U.S.A. 93: 10614-10619; Baldeschweiler e/ ⁇ /., 1995, PCT application WO95/2511 16; Shalon, et ai, 1995, PCT application WO95/35505: Heller er a/., 1997, Proc. Natl. Acad. ScL U.S.A.
  • high throughput screening can he used to measure the effects of RNAi's on complex molecular events such as signal transduction pathways, as well as cell functions including, but not limited to, cell function, apoptosis, cell division, cell adhesion, locomotion, exocytosis, and cell-cell communication.
  • Multicolor fluorescence permits multiple targets and cell processes to be assayed in a single screen. Cross- correlation of cellular responses will yield a wealth of information required for target validation and lead optimization.
  • the present invention provides a method for analyzing cells comprising providing an array of locations which contain multiple cells wherein the cells contain one or more fluorescent reporter molecules; scanning multiple cells in each of the locations containing cells to obtain fluorescent signals from the fluorescent reporter molecule in the cells; converting the fluorescent signals into digital data; and utilizing the digital data to determine the
  • a major component of the new drug discover ⁇ ' paradigm is a continually growing family of fluorescent and luminescent reagents that are used to measure the temporal arid spatial distribution, content, and activity of intracellular ions, metabolites, macromolecul ⁇ s, and organelles, Classes of these reagents include labeling reagents that measure the distribution arid amount of molecules in living and fixed cells, environmental indicators to report signal transduction events in time and space, and fluorescent protein biosensors to measure target molecular activities within living cells.
  • a multiparameter approach that combines several reagents in a single cell is a powerful new tool for drug discovery.
  • This method relies on the high affinity of fluorescent or luminescent molecules for specific cellular components.
  • the affinity for specific components is governed by physical forces such as ionic interactions, covalcnt bonding (which includes chimeric fusion with protein- based chromophores, fluorophores, and lumiphores), as well as hydrophobic interactions, electrical potential, and, in some cases, simple entrapment within a cellular component.
  • the luminescent probes can be small molecules, labeled macromolecul ⁇ s, or genetically engineered proteins, including, but not limited to green fluorescent protein chimeras.
  • fluorescent reporter molecules that can be used in the present invention, including, but not limited to, fluorescentiy labeled biomolccuics such as proteins, phospholipids, RNA and DNA hybridizing probes.
  • fluorescent reagents specifically synthesized with particular chemical properties of binding or association have been used as fluorescent reporter molecules (Barak et al., (1997), J. Biol. Chem. 272:27497-27500; Southwick el al, (1990), Cytometry 11:418-430; Tsien (1989) in Methods in Cell Biology, Vol. 29 Taylor and Wang (eds.), pp. 127-156).
  • Fluorescently labeled antibodies are particularly useful reporter molecules due to their high degree of specificity for attaching to a single molecular target in a mixture of molecules as complex as a cell or tissue,
  • the luminescent probes can be synthesized within the living cell or can be transported into the cell via several non-mechanical modes including diffusion, facilitated or active transport, signal-sequence-mediated transport, and endocytotic or pinocytotic uptake.
  • Mechanical bulk loading methods which are well known in the art, can also be used to load luminescent probes into living cells (Barber et al. (1996), Neuroscience Letters 207:17-20; Bright et al. (1996), Cytometry 24:226-233; McNeil (1989) in Methods in Cell Biology, Vol. 29, Taylor and Wang (eds.), pp. 153-173).
  • cells can be genetically engineered Io express reporter molecules, such as GFP, coupled to an IiNAi or probes of interest.
  • the luminescent probes Once in the cell, the luminescent probes accumulate at their target domain as a result of specific and high affinity interactions with the target domain or other modes of molecular targeting such as signal-sequence-mediated transport.
  • Fluorescently labeled reporter molecules are useful for determining the location, amount and chemical environment of the reporter. For example, whether the reporter is in a lipophilic membrane environment or in a more aqueous environment can be determined (Giuliano et al. (1995), Ann. Rev, of Biophysics and
  • sampling of sample materials may be accomplished with a plurality of steps, which include withdrawing a sample from a sample container and delivering at least a portion of the withdrawn sample to test cell culture (e.g., a cell culture wherein gene expression is regulated). Sampling may also include additional steps, particularly and preferably, sample preparation steps.
  • sample preparation steps may be included in one approach.
  • only one sample is withdrawn into the auto-sampler probe at a time and only one sample resides in the probe at one time,
  • multiple samples may be drawn into the auto-sampler probe separated by solvents, (n still other embodiments, multiple probes may be used in parallel for auto sampling.
  • sampling can be effected manually, in a semi-automatic manner or in an automatic manner.
  • a sample can be withdrawn from a sample container manually, for example, with a pipette or with a syringe-type manual probe, and then manually delivered Io a loading port or an injection port of a characterization system.
  • some aspect of the protocol is effected automatically (e.g., deliver ⁇ ' ), but some other aspect requires manual intervention (e.g., withdrawal of samples from a process control line).
  • the sample(s) are withdrawn from a sample container and delivered to the characterization system, in a fully automated manner—for example, with an auto-sampler.
  • auto-sampling may be done using a microprocessor controlling an automated system (e.g., a robot arm),
  • the microprocessor is user-programmable to accommodate libraries of samples having varying arrangements of samples (e.g., square arrays with "n-rows” by “n-columns,” rectangular arrays with “n-rows” by “m-columns,” round arrays, triangular arrays with “r ⁇ ” by “r-” by “r-” equilateral sides, triangular arrays with "r-base” by "s-” by “s-” isosceles sides, etc., where n, m, r, and s are integers).
  • Automated sampling of sample materials optionally may be effected with an auto- sampler having a healed injection probe (tip).
  • An example of one such auto sampler is disclosed in U.S. Pat. No. 6,175,409 Bl (incorporated by reference).
  • one or more systems, methods or both are used to identify a plurality of sample materials, Though manual or serai -automated systems and methods are possible, preferably an automated system or method is employed.
  • a variety of robotic or automatic systems are available for automatically or programmably providing predetermined motions for handling, contacting, dispensing, or otherwise manipulating materials in solid, fluid liquid or gas form according to a predetermined protocol.
  • Such systems may be adapted or augmented to include a variety of hardware, software or both to assist the systems in determining mechanical properties of materials.
  • Hardware and software for augmenting the robotic systems may include, but are not limited to, sensors, transducers, data acquisition and manipulation hardware, data acquisition and manipulation software and the like.
  • Exemplary robotic systems are commercially available from CAVRO Scientific Instruments (e.g., Model NO. RSP9652) or BioDot (Microdrop Model 3000).
  • the automated system includes a suitable protocol design and execution software that can be programmed with information such as synthesis, composition, location information or other information related to a library of materials positioned with respect to a substrate,
  • the protocol design and execution software is typically in communication with robot control software for controlling a robot or other automated apparatus or system.
  • the protocol design and execution software is also in communication with data acquisition hardware/software for collecting data from response measuring hardware. Once the data is collected in the database, analytical software may be used to analyze the data, and more specifically, Io determine properties of the candidate drugs, or the data may be analyzed manually,
  • Transfer of an exogenous nucleic acid into a host cell or organism can be assessed by directly detecting the presence of the nucleic acid in the cell or organism.
  • detection can be achieved by several methods well known in the art.
  • the presence of the exogenous nucleic acid can be detected by Southern blot or by a polymerase chain reaction (PCR) technique using primers that specifically amplify nucleotide sequences associated with the nucleic acid.
  • PCR polymerase chain reaction
  • Expression of the exogenous nucleic acids can also be measured using conventional methods.
  • mRJMA produced from an exogenous nucleic acid can be detected and quantified using a Northern blot and reverse transcription PCR (RT-PCR).
  • RNA from the exogenous nucleic acid can also be detected by measuring an enzymatic activity or a reporter protein activity.
  • siRNA activity can be measured indirectly as a decrease or increase in target nucleic acid expression as an indication that the exogenous nucleic acid is producing the effector RNA.
  • primers can be designed and used to amplify coding regions of the target genes. Initially, the most highly expressed coding region from each gene can be used to build a mode! control gene, although any coding or non coding region can be used. Each control gene is assembled by inserting each coding region between a reporter coding region and its ⁇ oly(A) signal.
  • plasmids would produce an mRNA with a reporter gene in the upstream portion of the gene and a potential RNAi target in the 3' non-coding region. The effectiveness of individual RNAi's would be assayed by modulation of the reporter gene.
  • Reporter genes useful in the methods of the present invention include acetohydroxy acid synthase (AHAS), alkaline phosphatase (AP), beta gaiactosidase (LacZ), beta glucuronidase (GIJS), chloramphenicol acetyltransferase (CAT), green fluorescent protein (GFP), red fluorescent protein (RFP), yellow fluorescent protein (YFP), cyan fluorescent protein (C FP), horseradish peroxidase (HRP), luciferase (Luc), nopaline synthase (NOS), octopine synthase (OCS), and derivatives thereof.
  • Multiple selectable markers are available that confer resistance to arapicilHn.
  • Methods to determine modulation of a reporter gene include, but are not limited to, fiuorametric methods (e.g. fluorescence spectroscopy,
  • Fluorescence Activated Cell Sorting FACS
  • fluorescence microscopy antibiotic resistance determination.
  • RNAi containing vector is transfected into appropriate cell lines which express that target nucleic acid.
  • Each selected RNAi construct is tested for its ability to modulate steady-state mRNA of the target nucleic acid,
  • any target niRNAs that "survive" the first round of testing are amplified by reverse Iranscriplase-PCR and sequenced (see, for example, Sambrook, J. et ai "Molecular Cloning: A Laboratory
  • RNAi constructs are analyzed to determine individual polymorphisms that allow mRNA to escape the current library of RNAi's. This information is used to further modify RNAi constructs to also target rarer polymorphisms.
  • RNAi vectors include, but are not limited to, elcctroporation, particle bombardment, microinjection, transfection with viral vectors, transfection with retrovirus-based vectors, and liposome-mediated transfection.
  • elcctroporation particle bombardment
  • microinjection microinjection
  • transfection with viral vectors transfection with retrovirus-based vectors
  • liposome-mediated transfection any of the types of nucleic acids that mediate RNA interference can be synthesized in vitro using a variety of methods well known in the art and inserted directly into a cell.
  • dsRNA and other molecules that mediate RNA interference are available from commercial vendors, such as Ribopharma AG (Kulmach, Germany), Eurogentec CSeraing, Belgium), Sequitur (Natick, Mass.) and Invitrogen (Carlsbad, Calif.).
  • Eurogentec offers dsllNA that has been labeled with fluorophores (e.g., HEX/ ' TET; 5 '-Fluorescein, 6-FAM; 3 '-Fluorescein, 6-FAM; Fluorescein dT internal; 5' TAMRA, Rhodamine; 3' TAMRA, Rjiodamine), which can also be used in the invention.
  • fluorophores e.g., HEX/ ' TET; 5 '-Fluorescein, 6-FAM; 3 '-Fluorescein, 6-FAM; Fluorescein dT internal; 5' TAMRA, Rhodamine; 3' TAMRA
  • RNAi molecules can be made through the well-known technique of solid-phase synthesis. Equipment for such synthesis is sold by several vendors including, for example, Applied Biosystems (Foster City, Calif.), Other methods for such synthesis that are known in the art can additionally or alternatively be employed. It is well-known Io use similar techniques to prepare oligonucleotides such as the phosphorothioat ⁇ s and alkylated derivatives.
  • RNA directly inserted into a cell can include modifications to cither the phosphate-sugar backbone or the nucleoside, For example, the phosphodiester linkages of natural RNA can be modified to include at least one of a nitrogen or sulfur heteroatom.
  • the interfering RNA can be produced enzymatically or by partial/total organic synthesis.
  • the constructs can be synthesized by a cellular RNA polymerase or a bacteriophage RNA polymerase (e.g., T3, T7, SP6). If synthesized chemically or by in vitro enzymatic synthesis, the RNA can be purified prior to introduction into a cell or animal.
  • IiNA can be purified from a mixture by extraction with a solvent or resin, precipitation, electrophoresis, chromatography or a
  • the interfering RNA construct can be used without, or with a minimum of purification to avoid losses due to sample processing.
  • the RNAi construct can be dried for storage or dissolved in an aqueous solution.
  • the solution can contain buffers or salts to promote annealing, and/or stabilization of the duplex strands.
  • buffers or salts thai can be used in the present invention include, but arc not limited to, saline, PBS, N-(2-Hydroxyethyl)piperazin- e-N'-(2-ethanesulfonic acid) (HEPESTM), 3-(N- Morpholino)propanesulfonic acid (MOPS), 2-bis(2-Hydroxycthylene)amino-2-(hydroxymclhyl)- 1,3-propaned- iol (bis-TRISTM), potassium phosphate (KP), sodium phosphate (NaP), dibasic sodium phosphate (Na 2 HPO-.), monobasic sodium phosphate OSIaHjPO 4 ), monobasic sodium potassium phosphate (NaKBlPO 4 ), magnesium phosphate (Mg 3 (PO 4 )2-4H 2 Q), potassium acetate (CH3COOH).
  • D(+)- ⁇ -sodium glycerophosphate (HOCH 2 CH(OH)CH 2 OPO 3 Na 2 ) and other physiologic buffers known to those skilled in the art.
  • Additional buffers for use in the invention include, a salt M-X dissolved in aqueous solution, association, or dissociation products thereof, where M is an alkali metal (e.g.. Li + , Na " , K + , Rb 1" ), suitably sodium or potassium, and where X is an anion selected from the group consisting of phosphate, acetate, bicarbonate, sulfate, pyruvate, and an organic monophosphate ester, glucose 6-phosphate or DL- ⁇ -glycerol phosphate.
  • M an alkali metal
  • X is an anion selected from the group consisting of phosphate, acetate, bicarbonate, sulfate, pyruvate, and an organic monophosphate ester, glucose 6-phosphate or DL- ⁇
  • the invention also includes pharmaceutical compositions containing nucleic acid conjugates.
  • the compositions arc suitable for internal use and include an effective amount of a pharmacologically active conjugate of the invention, alone or in
  • conjugates are especially useful in that they have very low, if any toxicity.
  • compositions of the invention can be used to treat, prevent, diagnose or image a pathology, such as a disease or disorder, or alleviate the symptoms of such disease or disorder in a patient.
  • compositions of the invention can be used to treat, prevent, diagnose or image a pathology associated with inflammation.
  • Compositions of the invention are useful for administration to a subject suffering from, or predisposed to, a disease or disorder which is related to or derived from a target to which the aptamers specifically bind or to the
  • RNAi's polynucleotides which the aptamcr-dclivered RNAi's are targeted to.
  • compositions of the invention can be used in a method for treating a patient having a pathology, e.g. cancer.
  • the method involves administering to the patient a composition comprising aptamers-RNAi's that bind a target (e.g., a protein), so that the RNAi is specifically delivered Io a target cell of choice and altering the biological function of the target, thereby treating the pathology.
  • a target e.g., a protein
  • the patient having a pathology e.g. the patient treated by the methods of this invention can be a mammal, or more particularly, a human.
  • conjugate or multi-domain molecules e.g., aptatner-RNAi's
  • aptatner-RNAi's are administered in amounts which will be sufficient to exert their desired biological activity.
  • compositions of the invention can be used in a method for inducing or enhancing immunogenicity of a target cell hi vitro or in vivo and modulating an immune response in patient comprising: obtaining a composition comprising at least one aptamer conjugated to at least one oligonucleotide molecule wherein the aptamer is specific for a desired target cell and the oligonucleotide is specific for a molecule associated with at least one factor associated with a nonsense mediated decay pathway (NMD); and, administering the composition in a
  • target cells comprise: a tumor cell, an infected cell, a tissue specific cell, an adipocyte, a stem cell, an immune cell, an organ specific cell or a transformed cell.
  • an antigen specific immune cell is optionally co- stimulated comprising administering to a patient co-stimulalory inducing agent is optionally administered to the patient.
  • the immune cells are specific for the novel antigens induced by the modulation of the NMD pathways, as well as any other antigen expressed by an abnormal cell, for example, PSMA.
  • an immune cell co-stimulatory agent targets one or more molecules comprising: 4- IBB (CDl 37), B7-1/2, 4-1BBL, OX40L, CD40, LIGHT, OX40, CD2, CD3.
  • HVEM TNFRSF14, ATAR, LIGHTR, TR2
  • CD150 SLAM
  • CD152 CTLA-4
  • CD 154 CD40L
  • CD 178 FasL
  • CD209 DC-SIGN
  • CD 270, CD277 AITR, AITRL
  • B7-H3, B7-H4, BTLA HLA-ABC
  • HLA-DR HLA-DR
  • ICOS ICOSL
  • B7RP-1 NKG2D
  • PD-I CD279
  • PD-Ll B7-H1
  • PD-L2 B7-DC
  • TCR- ⁇ TCR- ⁇ , TCR- ⁇ , TCR- ⁇ , TCR- ⁇ .
  • ZAP-70 lymphotoxin receptor
  • immune cells comprise T cells (T lymphocytes), B cells (B lymphocytes), antigen presenting cells, dendritic cells, monocytes, macrophages, myeloid suppressor cells, natural killer (NK) cells, NK T cells, suppressor cells, T regulatory cells (Tregs), cytotoxic T lymphocytes (CTLs), CTL lines, CTL clones, CTLs from tumor, inflammatory, or oilier infiltrates and subsets thereof.
  • One aspect of the invention comprises a pharmaceutical composition of the invention in combination with other treatments for inflammatory and autoimmune diseases, cancer, and other related disorders.
  • the pharmaceutical compositions of the invention may contain, for example, more than one aptamcr-RNAi.
  • a pharmaceutical composition of the invention, containing one or more compounds of the invention is administered in combination with another useful composition such as an anti-inflammatory agent, an imrnuno stimulator, a cheniotherapeutic agent, an antiviral agent, or the like.
  • the compositions of the invention may be administered in combination with a cytotoxic, cytostatic, or cheniotherapeutic agent such as an alkylating agent, anti-metabolite, mitotic inhibitor or cytotoxic antibiotic, as described above.
  • the currently available dosage forms of the known therapeutic agents for use in such combinations will be suitable.
  • Combination therapy includes the administration of an aplamer-RNAi conjugate of the invention and at least a second agent as part of a specific treatment regimen intended to provide the beneficial effect from the co-action of these therapeutic agents.
  • the beneficial effect of the combination includes, but is not limited to, pharmacokinetic or pharmacodynamic co-action resulting from the combination of therapeutic agents.
  • Administration of these therapeutic agents in combination typically is carried out over a defined time period (usually minutes, hours, days or weeks depending upon the combination selected).
  • Combination therapy may, but generally is not, intended to encompass the administration of two or more of these therapeutic agents as part of separate monotherapy regimens that incidentally and arbitrarily result in the combinations of the present invention.
  • Combination therapy is intended to embrace administration of these therapeutic agents in a sequential manner, that is, wherein each therapeutic agent is administered at a different time, as well as administration of these therapeutic agents, or at leas! two of the therapeutic agents, in a substantially simultaneous manner. Substantially simultaneous administration can be
  • Sequential or substantially simultaneous administration of each therapeutic agent can be effected by any appropriate route including, but not limited to, topical routes, oral routes, intravenous routes, intramuscular routes, and direct absorption through mucous membrane tissues.
  • the therapeutic agents can be administered by the same route or by different routes.
  • a first therapeutic agent of the combination selected may be administered by injection while the other therapeutic agents of the combination may be administered topically.
  • all therapeutic agents may be administered topically or all therapeutic agents may be administered by injection.
  • the sequence in which the therapeutic agents are administered is not narrowly critical unless noted otherwise.
  • Combination therapy also can embrace the administration of the therapeutic agents as described above in further combination with other biologically active ingredients.
  • the combination therapy further comprises a non-drug treatment
  • the non-drug treatment may be conducted at any suitable time so long as a beneficial effect from the co-action of the combination of the therapeutic agents and non-drug treatment is achieved. For example, in appropriate cases, the beneficial effect is still achieved when the non-drug treatment is temporally removed from the administration of the therapeutic agents, perhaps by days or even weeks.
  • Therapeutic or pharmacological compositions of the present invention will generally comprise an effective amount of the active component(s) of the therapy, dissolved or dispersed in a pharmaceutically acceptable medium.
  • Pharmaceutically acceptable media or carriers include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Supplementary active ingredients can also be incorporated into the therapeutic compositions of the present invention.
  • the therapeutically effective amount or dose can be estimated initially from activity assays in cell cultures and/or animals.
  • a dose can be formulated in animal models to achieve a circulating concentration range that includes the IC 50 as determined by activity assays (e,g., the concentration of the test compound, which achieves a half-maximal inhibition of the proliferation activity). Such information can be used to more accurately determine useful doses in humans.
  • Toxicity and therapeutic efficacy of the peptides described herein can be determined by standard pharmaceutical procedures in experimental animals, e.g., by determining the IC 50 and the LD50 (lethal dose causing death in 50% of the tested animals) for a subject compound. The data obtained from these activity assays and animal studies can be used in formulating a range of dosage for use in human.
  • the dosage may vary depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (Sec e.g., Fingl, et ah, 1975, in "The Pha ⁇ nacoiogical Basis of Therapeutics", Ch. 1 p.l). Dosage amount and interval may be adjusted individually to provide plasma levels of the active moiety which are sufficient to maintain therapeutic effects, termed the minima] effective concentration (MEC).
  • MEC minima effective concentration
  • the MEC will vary for each preparation, but can be estimated from in vitro and/or in vivo data, e.g., the concentration necessary to achieve 50-90% inhibition of a proliferation of certain cells may be ascertained using the assays described herein. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. HPLC assays or bioassays can be used to determine plasma concentrations. Dosage intervals can also be determined using the MEC value. Preparations should be administered using a regimen, which maintains plasma levels above the MEC for 10-90% of the time, preferable between 30-90% and most preferably 50-90%.
  • dosing can also be a single administration of a slow release composition described h ⁇ reinabove, with course of treatment lasting from several days to several w r e ⁇ ks or until cure is effected or diminution of the disease state is achieved,
  • the amount of a composition to be administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of
  • compositions will be known to those of skill in the art in light of the present disclosure.
  • such compositions may be prepared as mjectables, cither as liquid solutions or suspensions: solid forms suitable for solution in, or suspension in. liquid prior to injection; as tablets or other solids for oral administration; as time release capsules; or in any other form currently used, including eye drops, creams, lotions, salves, inhalants and the like.
  • sterile formulations such as saline -based washes, by surgeons, physicians or health care workers to treat a particular area in the operating field may also be particularly useful.
  • Compositions may also be delivered via microdcvice, microparticle or other known methods.
  • therapeutics will be administered in a manner compatible with the dosage formulation, and in such amount as is pharmacologically effective.
  • the formulations arc easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed.
  • the quantity of active ingredient and volume of composition to be administered depends on the host animal to be treated. Precise amounts of active compound required for administration depend on the judgment of the practitioner and arc peculiar to each individual.
  • a minimal volume of a composition required to disperse the active compounds is typically utilized. Suitable regimes for administration are also variable, but would be typified by initially administering the compound and monitoring the results and then giving further controlled doses at further intervals.
  • the active drug component can be combined with an oral, non-toxic, pharmaceutically acceptable inert carrier such as ethanol, glycerol, water and the like.
  • an oral, non-toxic, pharmaceutically acceptable inert carrier such as ethanol, glycerol, water and the like.
  • suitable binders, lubricants, disintegrating agents, and coloring agents can also be incorporated into the mixture.
  • suitable binders include starch, magnesium aluminum silicate, starch paste, gelatin, melhylcellulose. sodium carboxymethylcellulose and/or
  • Lubricants used in these dosage forms include sodium oleate, sodium stearate, magnesium stearate, sodium bcnzoate, sodium acetate, sodium chloride, silica, talcum, stearic acid, its magnesium or calcium salt and/or polyethyleneglycoi, and the like.
  • - /3 - include, without limitation, starch, methyl cellulose, agar, bentonite, xanthan gum starches, agar, alginic acid or its sodium salt, or effervescent mixtures, and the like.
  • Diluents include, e.g., lactose, dextrose, sucrose, niaruiitol, sorbitol, cellulose and/or glycine.
  • compositions of the invention can also be administered in such oral dosage forms as timed release and sustained release tablets or capsules, pills, powders, granules, elixirs, tinctures, suspensions, syrups and emulsions.
  • Suppositories are advantageously prepared from fatty emulsions or suspensions.
  • compositions may be sterilized and/or contain adjuvants, such as preserving, stabilizing, wetting or emulsifying agents, solution promoters, salts for regulating the osmotic pressure and/or buffers. In addition, they may also contain other therapeutically valuable substances.
  • adjuvants such as preserving, stabilizing, wetting or emulsifying agents, solution promoters, salts for regulating the osmotic pressure and/or buffers.
  • adjuvants such as preserving, stabilizing, wetting or emulsifying agents, solution promoters, salts for regulating the osmotic pressure and/or buffers.
  • the compositions are prepared according to conventional mixing, granulating, or coating methods, and typically contain about 0.1% to 75%, preferably about 1% to 50%, of the active ingredient.
  • Liquid, particularly injectable compositions can, for example, be prepared by dissolving, dispersing, etc.
  • the active compound is dissolved in or mixed with a pharmaceutically pure solvent such as, for example, water, saline, aqueous dextrose, glycerol, ethanoi, and the like, to thereby form the injectable solution or suspension.
  • a pharmaceutically pure solvent such as, for example, water, saline, aqueous dextrose, glycerol, ethanoi, and the like.
  • solid forms suitable for dissolving in liquid prior to injection can be formulated.
  • compositions of the present invention can be administered in intravenous (both bolus and infusion), intraperitoneal, subcutaneous or intramuscular form, all using forms well known to those of ordinary skill in the pharmaceutical arts.
  • injectables can be prepared in conventional forms, cither as liquid solutions or suspensions.
  • Parenteral injectable administration is generally used for subcutaneous, intramuscular or intravenous injections and infusions. Additionally, one approach for parenteral administration employs the implantation of a slow-release or sustained-released systems, which assures that a constant level of dosage is maintained, according to U.S. Pat. No. 3,710,795, incorporated herein by reference.
  • compositions for the present invention can be administered in intranasal form via topical use of suitable intranasal vehicles, inhalants, or via transdermal
  • the dosage is administered in the form of a transdermal delivery system, using those forms of transdermal skin patches well known to those of ordinary skill in that art.
  • the dosage is administered in the form of a transdermal delivery system, the dosage
  • Topical preparations include creams, ointments, lotions, aerosol sprays and gels, wherein the concentration of active ingredient would typically range from 0.01 % to 15%, w/ ' w or w/ ' v.
  • cxcipients include pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like.
  • the active compound defined above may be also formulated as suppositories, using for example, polyalkylene glycols, for example, propylene glycol, as the carrier.
  • suppositories are advantageously prepared from fatty emulsions or suspensions.
  • Liposome delivery systems such as small unilamellar vesicles, large unilamellar vesicles and multilamellar vesicles.
  • Liposomes can be formed from a variety of phospholipids, containing cholesterol, stearylamine or phosphatidylcholines.
  • a film of lipid components is hydrated with an aqueous solution of drug to a form lipid layer encapsulating the drug, as described in U.S. Pat. No. 5,262,564.
  • the aptamer molecules described herein can be provided as a complex with a lipophilic compound or non-immunogemc, high molecular weight compound constructed using methods known in the art.
  • a lipophilic compound or non-immunogemc high molecular weight compound constructed using methods known in the art.
  • nucleic-acid associated complexes is provided in U.S. Pat. No. 6,011,020.
  • the compounds of the present invention may also be coupled with soluble polymers as targetable drug carriers.
  • soluble polymers can include polyvinylpyrrolidone, pyran copolymer, polyhydroxypropyl-methacrylamide-phenol, polyhydroxyethylaspanamidephenol, or
  • the compounds of the present invention may be coupled to a class of biodegradable polymers useful in achieving controlled release of a drug, for example, poiy lactic acid, polyepsilon caprolactone, poly hydroxy butyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates and cross- linked or ampl ⁇ pathic block copolymers of hydrogels.
  • biodegradable polymers useful in achieving controlled release of a drug, for example, poiy lactic acid, polyepsilon caprolactone, poly hydroxy butyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates and cross- linked or ampl ⁇ pathic block copolymers of hydrogels.
  • the pharmaceutical composition to be administered may also contain minor amounts of non-loxic auxiliary substances such as wetting or emulsifying agents, ptl buffering agents, and other substances such as for example, sodium acetate, and trielhanolamine oleate.
  • auxiliary substances such as wetting or emulsifying agents, ptl buffering agents, and other substances such as for example, sodium acetate, and trielhanolamine oleate.
  • the dosage regimen utilizing the aptamer-RNAi's is selected in accordance with a variety of factors including type, species, age, weight, sex and medical condition of the patient; the severity of the condition to be treated; the route of administration; the renal and hepatic function of the patient; and the particular ap tamer or salt thereof employed.
  • An ordinarily skilled physician or veterinarian can readily determine and prescribe the effective amount of the drug required to prevent, counter or arrest the progress of die condition,
  • Oral dosages of the present invention when used for the indicated effects, will range between about 0.05 to 7500 mg/day orally.
  • the compositions are preferably provided in the form of scored tablets containing 0.5, 1.0. 2.5, 5.0. 10.0. 15.0. 25.0, 50.0, 100.0, 250,0, 500.0 and 1000.0 mg of active ingredient.
  • Infused dosages, intranasal dosages and transdermal dosages will range between 0,05 to 7500 mg/day.
  • Subcutaneous, intravenous and intraperitoneal dosages will range between 0.05 to 3800 mg/day.
  • Effective plasma levels of the compounds of the present invention range from 0.002 mg/mL to 50 mg/niL.
  • Compounds of the present invention may be administered in a single daily dose, or the total daily dosage may be administered in divided doses of two, three or four times daily.
  • RNAi can also be produced through intramolecular hybridization of complementary- regions within a single RNA molecule.
  • An expression unit for synthesis of such a molecule comprises the following elements, positioned from left to right: 1. A DNA region comprising a viral enhancer; 2. A DNA region comprising an immediate early or early viral promoter oriented in a 5' to 3' direction so that a DNA segment inserted into the region of part 4 is transcribed; 3. A DNA region into which a DNA segment can be inserted. Preferably this region contains at least one restriction enzyme site; 4. A DNA region comprising a transcriptional terminator arranged
  • kits for targeting nucleic acid sequences of cells and molecules associated with modulation of the immune response in the treatment of diseases such as, for example, infectious disease organisms, cancer, autoimmune diseases and the like.
  • the kits can be used to target any desired nucleic sequence and as such, have many applications,
  • a kit comprises: (a) an aptamer-RNAi that targets a desired cell and nucleic acid sequence, and (b) instructions to administer to cells or an individual a
  • the kit may comprise pharmaceutically acceptable salts or solutions for administering the aptamer-RNAi.
  • the kit can further comprise instructions for suitable operational parameters in the form of a label or a separate insert.
  • the kit may have standard instructions informing a physician or laboratory technician to prepare a dose of aptamer-RNAi.
  • the kit may further comprise a standard or control information so that a patient sample can be compared with the control information standard to determine if the test amount of an aptarner-RNAi is a therapeutic amount consistent with for example, a shrinking of a tumor or decrease in viral load in a patient.
  • Embodiments of the invention may be practiced without the theoretical aspects presented. Moreover, the theoretical aspects are presented with the understanding that Applicants do not seek to be bound by the theory presented.
  • NMD inhibition-induced expression of novel antigens abrogated tumor growth. Given that in this experiment NMD was inhibited in all cells from day zero, from a clinical standpoint - involving targeted delivery of siRN A to tumor bearing individuals - the questions were whether expression of new antigens in an established tumor would suffice to inhibit tumor growth, and what proportion of tumor cells need to express new antigens to exert a therapeutic impact.
  • aptamers can be used to target siRNA to specific cells in vitro and in vivo.
  • the first objective is to construct aptamer- oligonucleotides fusion ODNs that target the siRN A to tumor cells leading to effective inhibition ofNMD.
  • Aptamers are directed to human PSMA and rat Hcr2/Neu.
  • siRNAs arc generated against murine SMGl, Upfl , UpG and UpO.
  • Aptamer-oligonucleotides fusions are generated using existing algorithms and exploring novel algorithms to maximize the function of the conjugated s ⁇ RNA.
  • the aptamer-oligonucleotides ODNs are conjugated to peptides which promote cytoplasmic translocation from endosomcs, such the HIV derived tat peptide, a fusogenic peptide from influenza hemagglutinin protein, a 9mer Arg oligopeptide and others, (n).
  • the aptamer-oligonucleotides chimeras are conjugated to cholesterol or polyethylene glycol.
  • aptamer-oligonucleotides are first screened for NMD inhibition using a standard assay based on rescuing the stable expression of mRN A from an NMD reporter plasmid encoding a glob in transcript with an engineered PTC that accumulates reduced levels of mRNA in transiently trans f ⁇ cted cells.
  • Promising aptamer-oligonucleotides are subjected to transcript expression profiling to determine their ability to upregulate multiple NMD substrates. In view of the physiological roles of NMD and other roles of SMGI and Upfl discussed above, effects on cell viability and proliferation arc closely monitored.
  • ODNs aptamer-oligonucleotides
  • pharmacokinetics studies are first carried out to determine the half-life and biodistribution of the ODNs as a function of dose, frequency and route of administration. Specificity of in vivo tumor targeting expressing the cognate receptor are determined and mice are monitored for adverse effects.
  • an in vivo NMD reporter system consisting of stably or transiently iransfecting PSMA-ex pressing tumor cell lines with an OVA gene containing a PTC upstream the H-2b-rcstrictcd dominant class 1 and class 11 epitopes (OVAPTC), and a downstream in iron.
  • OVAPTC H-2b-rcstrictcd dominant class 1 and class 11 epitopes
  • Targeting in vitro and in vivo the OVAPTC encoded PSM A-expressing tumor cells with a PSMA aplamer-oligonucieotides ODNs should stimulate class I and class II presentation that can be detected using OT-I and OT-II T cells, respectively.
  • Mechanistic studies - is inhibition of tumor growth due to enhanced tumor antigenicity. As discussed above, inhibition of NMD or its factors can have direct cytotoxic effect that could account for an observed tumor inhibition. To rule out a direct cytotoxic effect in vivo, and to demonstrate that tumor inhibition is immune-mediated, aptamcr-oligonucleotidcs mediated tumor inhibition are measured in nude mice and/or in mice depleted of CD4 + and CDS " T cells with antibodies.
  • thai enhancement of tumor immunity is mediated by the expression of novel antigenic determinants, and not "immunogenic death,'" the aptamer- oligonucleotidcs treated mice which rejected the tumor are challenged with NMD-inhibited tumors (tumor cells stably transduced with lenti vectors expressing siRNA targeted to NMD factors) and tumor growth is compared to that of non-NMD inhibited tumor challenge.
  • in vitro T cell assay are carried out against NMD-inhibited versus non-NMD inhibited tumor cell targets.
  • Tumor cells stably expressing SMG- I and Upf-2 NMD factor siRNAs under doxacycline control were generated by lenti viral transduction, When SMG-I or Upf-2 siRNAs are expressed (cells are cultured in the presence of drug) the corresponding RNAs are downregulatcd and NMD is inhibited, NMD inhibition in tumor cells expressing SMG-I or Upf-2 siRNA (mice receive doxacyline in the drinking water) abrogates tumor growth. Importantly, doxacyline- dcpcndcnt SMG-I or Upf-2 siRNA expression has no effect on the exponential growth of tumor cells in culture.
  • NMD was inhibited specifically in tumor cells resulting in tumor regression, conceivably as a result of uprcgulation of new antigenic products.
  • This approach is clinically feasible, from the standpoint of cost, access to reagents, and regulatory approval process, and broadly applicable to most if not all cancer patients.
  • Example 3 Induction of tumor Immunity by targeted inhibition of nonsense mediated inRNA decay
  • mice were implanted with 1 x 10 6 PSMA-CT26 tumor cells and injected with 400 pmolcs of aptamcr-siRNA in 100 ⁇ l PBS via the tail vein at days 3, 5, 7, 9, 1 1 and 13.
  • treatment with PSMA aptamer-siRNA was administered at days 5, 7, 9, 11 and 13, and a single dose of 500 pmolcs of 4- IBB aptamer dimer was administered on day e>.
  • mice 1030Oi T° monitor metastasis, C57BL/6 mice were implanted with 10 " B16-PSMA transduced cells by the tail vein and injected with 400 pmolcs of aptamer-siRNA conjugates at days 5, 8, 11. 14 and 17.
  • the mice were euthanized and their lungs were weighed.
  • GM-CSF- cxprcssing Bl 6/F10 tumor cells were irradiated (50 Gy) and 5 x 10 5 cells were injected subcutancously at days 1 , 4 and 7, or days 5, 8 and 1 1 as described previously (Quezada. S. A., et al. J. Clin. Invest, 116, 1935-1945 (2006)).
  • PSMA apiamer-siRNA conjugates The PSMA aptamer, 5'- GGGAGGACGAUGCGGAUCAGCCAUGUUUACGUCACUCCUUGUCAAUCCUCAUCGG CAGACGACUCGCCCGA-3 " (SEQ ID NO: 1) was cloned into pUC57 between Kpnl and Barn KI restriction sites. siRNAs were screened using the psiCHECK system (Proracga) from candidates generated by the HPCdispatcher and OpenBiosystetn algorithms.
  • the DNA template for the aptamcr-siRNA guide strand was generated by PCR amplification using forward primer 5 '-TAATACGACTCACTATAGGGAGGACGATGCGG-:) ' ⁇ SEQ ID NO: 2) and reverse primers 5 '-AAGCGTTATGTTTGGTGGAAGTCGGGCGAGTCGTCTG-S ' (SEQ ID NO: 3) for control siRNA, 5 '-AAGCCATGACTAACACTGA AATCGGGCGAGTCGTCTG-:) ' (SEQ ID NO: 4) Up/2 siRNA, and 5 " -AAAATTCTCCGAACGTGTCACTCGGGCGAGTCGTCTG- 3' (SEQ ID NO: 5) for Srngl siRNA, The PCR products were purified using the QIAprep Spin columns (Qiagcn) RNA was transcribed using the T7CY639F) polymerase and hybridized to the corresponding passenger strands (control siRNA sequence: 5'-
  • PSMA-expressitig CT26 minor cell lines.
  • the PSMA complementary DNA was PCR-amplificd using forward primer 5'-
  • Plasmid was transiently transfeeted into the Phoenix -AMPHO 293 packaging cell lines and viral supernatant was used to transduce CT26 colon carcinoma (H-2 d ) and B16/F 10 melanoma (H-2 b ) tumor cell lines.
  • PSMA-expressing cells were isolated by cell sorting using PSMA-PE labeled anti-PSMA antibody from MBL,
  • Double-stranded oligonucleotides corresponding to the guide and passenger strands of Smgl, Upf2 or control siRNA modified to contain overhangs compatible with BgIU and Kpnl restriction sites were cloned into the BgIII and Kpnl sites of pFRT-U ⁇ tctQ plasmid.
  • the U ⁇ tetO-shRNA cassettes from the pFRT piasmids were isolated by PCIi (forward primer: 5'- GATCAGCGGCCGCTGCAGAAGGTCGGGCAGGAAGAG-3 " (SEQ ID NO: I I); reverse primer: S'-GTTAAGCATGCCCACACTGGACTAGTGGATC-S (SEQ ID NO: 12) and cloned into the Notl/Sphl restriction sites of PTIG icntiviral vector to generate pTlG-U ⁇ tetQsiiRNA plasn ⁇ ds, pTIG-U ⁇ tetQshRNA DNA was cotransfected into 293T cells with lentiviral packaging piasmids pCHPG-2, pCMV-rcv and PC MV -gag and lentivirus-containing supernatant was collected and concentrated by ccntrifugation.
  • PCIi forward primer: 5'- GATCAGCGGCCGCTGCAGAAGG
  • CT26 colon carcinoma (H-2 ⁇ ) and B 16/FI G melanoma (H ⁇ 2°) tumor cell lines were infected with lentiviral vectors and stably transduced GFP-cxprcssing ceils were isolated by sorting.
  • shRNA oligonucleotides used were as follows, Control shRNAs: 5'- GATCAATTCTCCGAACGTGTCACTTCCTGACCCAAAGTGACACGTTCGGAGAATTTT TTTGTAC-3' (SEQ ID NO: 13): 5 ' - AAAAAAATTCTCCGAACGTGTCACTGGGTCAGGAAGTGACACGTTCGGAGAATT-S'
  • BG, BGPTC and OVA-BG PTC transduced cells ⁇ -globin: forward, 5'-ACCACCGTAGAACGCAGATCG-3' (SEQ ID IsIO: 28); reverse, 5 '-CCTGAACTTCTCAGGATCC-J ' (SEQ ID NO: 29).
  • mice C57BL/6 mice (CD45.2; Thy 1.2) were implanted subeutaneously with 5 x 10 4 B 16 tumor cells and 8 days after tumor inoculation 5 x 10° pcptide-activatcd OT-I (CD45.1 ) or Pmcl-1 CD8 " T cells were injected intravenously via the tail vein, At the same day the drinking water was supplemented with 10% sucrose (Sigma) and with or without 2 mg ml "1 doxycycline (Sigma). At day 14 after tumor implantation mice were euthanized, tumors removed and mechanically disaggregated by collagenasc treatment (400U ml " l ).
  • tumors were surgically removed, cells dispersed by incubation with 400 U ml ' 1 of collagcnasc, washed three times with PBS, and cell-associated ' 2 P was measured in a scintillation counter.
  • U ⁇ tetGshRNA transduced CT26 tumor cells were implanted subcutancously in Balb/ ' c or Nude mice. At the day of tumor implantation mice started receiving water supplemented with 10% sucrose with or without 2 mg ml "1 doxycycline (Sigma).
  • mice were implanted with 1 x 10 h P8MA-CT26 tumor cells and injected with 400 pmoles of aptamer-siRNA in 100 ml PBS via the tail vein at days 3, 5, 7, 9, 11 and 13.
  • treatment with PSMA aptamer-siRNA was administered at days 5, 7, 9, 1 1 and 13, and a single dose of 500 pmoles of 4-1BB aptamcr dimcr was administered on day 6.
  • mice were implanted with 10 5 B16-PSMA transduced cells via the tail vein and injected with 400 pmoles of aptamer-siRNA conjugates at days 5, 8, 11, 14 and 17.
  • the mice were euthanized and their lungs were weighed.
  • GM-CSF- expressing B16/F10 tumor cells were irradiated (50 Gy) and 5 ⁇ IQ ⁇ cells were injected subcutaneously at days 1, 4 and 1 , or days 5, 8 and 1 1.
  • NTD Nonsense mediated mRNA decay
  • shRNAs NMD factor short hairpin RNAs
  • CT26 colon carcinoma tumor cells were transduced with a lentiviral vector (PTTG-U6letOshRNA) encoding Smgl or UpP shRNAs expressed from a tet-regulated U6 promoter (Aagaard, L. et a!. MoI. Ther. 15, 938-945 (2007)).
  • slxRNA expression can be upregulated in vitro by adding doxycycline to the culture medium and hi vivo by providing doxycycline in the drinking water.
  • B16/F10 tumor cells harboring the doxycycline-inducible Smgl, UpJl, and control shRNAs were stably transfected with an NMD reporter plasmid encoding the dominant MHC class 1 epitope of the chicken ovalbumin gene (OVA) upstream of a PTC (Diagrams in Figure 4A and Figure 8A), Tumor-bearing mice were infused with OT-I transgenic CD8 " T cells which recognize the OVA MHC class I-rcstricted epitope, or with Pmcl-1 transgenic CD8 " T cells which recognize an MFIC class I-restricted epitope in the endogenous gpl 00 tumor antigen expressed in B16 tumor cells 19, gpl 00 expression is not under NMD control, As shown in Figure 4 A, unlike Pmei-1 T cells, the OT-I T cells failed to accumulate to significant levels in the OVA negative B 1 ⁇ /F i O tumors or in tumors transfectcd with the PTC
  • FIG. 4B shows that tumor cells expressing Smgl or Up/2, but not control.
  • shRNA grew initially but failed to progress. Tumor inhibition was immune-mediated because the tumors grew in nude mice ( Figure 4C), and mice which rejected the tumors shown in Figure 4B, but not age-matched control mice, resisted a second challenge with parental tumor cells. Delaying doxycycline treatment of mice expressing SMG-I shRNA diminished the tumor inhibitory impact which was completely lost when drag treatment was delayed for six days (Figure 9). Tumor rejection correlated with the induction of T cell responses against tumor cells expressing Smgl shRNA.
  • NMD factor siRNAs were targeted to tumor cells using oligonucleotide aptarner ligands (Gold, L. J. Biol Cheni. 270, 13581 -- 13584 (1995); Nimjce, S. M., et al Anriu. Rev. Med. 56, 555-583 (2005)).
  • PSMA conjugated siRNA Io an oligonucleotide aptamer which binds to prostate specific membrane antigen ( PSMA) as shown in Figure 1 1.
  • PSMA expressing CT26 and B16 tumor cell lines were generated by transduction with a PSMA encoding expression vector, and expression of PSM A was confirmed by flow cytometry.
  • PSMA-CT26 tumor bearing mice were treated with PSMA aptamcr- Smgl siRNA and an agonistic 4-1 BB aptamer dimer (McNamara, J. O. et at. J. CHn. Invest. 118, 376-386 (2008)).
  • the stringency of NMD inhibition and 4-1 BB costimulation was adjusted to elicit a limited antitumor effect when applied separately by delaying treatment with PSMA aptamer-siRNA conjugates from day 3 to day 5 and administering a single dose of 4- IBB aptamer on day 6.
  • combination therapy with PSMA a ⁇ tamer-S>?jg/ siRNA and 4- IBB aptamer was synergistic.
  • mice were implanted in opposite flanks with PSMA- expressing and parental CT26 tumor cells and PSMA aptamer conjugated to control or Smgl siRNA was administered systemically by tail vein injection (Figure 6A).
  • Figure 6B shows that ' " P-labeled PSMA aptamer- Smgl si UNA conjugate accumulated preferentially in PSMA- cxprcssing tumor cells.
  • Figure ⁇ C shows that systemic administration of PSMA aptamer conjugated SmgL but not control, siRNA inhibited the growth of PSMA-expressing CT26 tumor cells but not the contralateral ⁇ implanted parental CT26 tumor cells.
  • Figure 15 shows a snapshot of the tumor-bearing mice at the day of sacrifice.
  • Tumor targeted NMD inhibition is a novel approach to stimulate protective antitumor immunity. Instead of stimulating or potentiating immune responses against existing, often weak, antigens expressed in the tumor cells, the goal of current tumor vaccination protocols, NMD inhibition generates novel antigenic determinants in situ in the disseminated tumor lesions. It should be noted that NMD control of gene expression is "leaky"'. In addition to the first round of translation, known as pioneer translation, the efficiency of nonsense-mediated degradation varies among individual mRN A targets, Immune recognition is, therefore, a consequence of uprcgulation of NMD controlled products above a certain threshold that was set by the natural immune tolerance mechanisms.
  • the NMD inhibition strategy described in this study consists of a single reagent that can be synthesized in a cell-free chemical process; it obviates the need to identify TRAs or adjuvants, and is broadly applicable as it targets a common pathway in all tumors.
  • the potency of the NMD inhibition approach was sevidenced when compared to GVAX vaccination, a "gold standard' * best-in-class vaccination protocol.
  • this first generation aplamer-siRNA conjugates and the dose arid treatment schedule can be further optimized, It would be of interest to dete ⁇ nine in future studies whether the NMD-induced antigens are cross- reactive among different tumors, identify the dominant antigens induced by NMD inhibition, and whether "epitope spread' 1 to constitulively expressed tumor antigens contributes to protective immunity.
  • Truncated products generated by PTCs are not good substrates for generating novel antigenic determinants because the normal expression of the non-truncated products in the absence of a PTC will have triggered tolerance. Yet, nonsense mutations generating PTCs are rare events and it is unlikely that the NMD system has evolved to counter their potential deleterious effects.
  • NMD nuclear deoxyribonucleic acid
  • Such products will encode novel peptides and hence could provide antigenic determinants to which the immune system has not be tolerized.
  • an important role of NMD is to maintain splicing integrity. The efficiency and accuracy of splicing is notoriously imperfect.
  • Such transcripts, encoding novel peptides corresponding to intron sequences will often contain PTCs and hence become targets for NMD elimination ⁇ Behm-Ansmant, I. et al. FEBS Lett 581, 2845-2853 (2007); Isken, O. & Maquat, L. E. Nat Rev Genet 9. 699:712 (2008)).
  • NMD is also responsible for the elimination of transcripts encoding nonproductively rearranged T cell receptors and immunoglobulin chain.
  • a significant proportion of gene products (>15%) that are upregulated when NMD is inhibited, such as by targeting Upf-1 with siRNA, are involved in amino acid biosynthesis and transcription factors which coordinate cellular responses to starvation. Since starvation also downregulates translation thru phosphorylation and inhibition of eIF2 ⁇ , which in turn inhibits NMD efficiency, it appears that the response to starvation is in part under NMD control.
  • NMD is also implicated in several instances of products autoregulating alternative splicing (e.g., serine-arginine (SR)-rich proteins and hnRNP splicing factors such as SC35, calpain, C DC-like kinases), biosynthesis of selenoproteins, and telomere synthesis (Holbrook, J, A., et at. Nat Gene! 36, 801 -808 (2004); Iskcn. O. & Maquat, L . E. Nat Rev Genet 9, 699:712 (2008)).
  • SR serine-arginine
  • hnRNP splicing factors such as SC35, calpain, C DC-like kinases
  • Such products could provide a source of tumor-specific antigenic determinants downstream of the recombination site. Consistent with this hypothesis, decreased immune infiltrate are seen in tumors with MSl phenotype which correlates with increased levels of Upf-1 in the tumors. Inhibiting NMD will further augment the production of such turn or- specific antigens.

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Abstract

L'invention porte sur des compositions d'induction ou d'augmentation de l'antigénicité d'une cellule cible par modulation de la voie de dégradation à médiation par un non-sens dans la cellule cible. Les compositions comprennent un ou plusieurs aptamères fournissant une spécificité et une distribution d'un oligonucléotide à la cible. Ces compositions ont une large applicabilité dans le traitement de nombreuses maladies.
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US8603814B2 (en) 2009-07-20 2013-12-10 Rutgers The State University Of New Jersey Method of inhibiting nonsense-mediated mRNA decay
US9849146B2 (en) 2009-07-20 2017-12-26 Rutgers, The State University Of New Jersey Inhibition of nonsense mediated mRNA decay by drugs that prevent hypusination of eukaryotic initiation factor 5A
WO2012174126A1 (fr) * 2011-06-13 2012-12-20 Universyty Of Medicine And Dentistry Of New Jesey Procédé d'inhibition de la dégradation des arnm induite par des codons d'arrêt
JP6120848B2 (ja) 2011-08-15 2017-04-26 メディミューン，エルエルシー 抗ｂ７−ｈ４抗体およびその使用
AU2013361275B2 (en) 2012-12-19 2016-11-24 Amplimmune, Inc. Anti-human B7-H4 antibodies and their uses
PL3041958T4 (pl) 2013-09-04 2020-11-02 Cold Spring Harbor Laboratory Redukcja rozpadu mRNA mediowanego sekwencjami nonsensownymi
US9926565B2 (en) * 2014-09-23 2018-03-27 Oregon Health & Science University Aptamer-RNAi therapeutic compositions
CA2963923A1 (fr) 2014-10-08 2016-04-14 Pacific Northwest Research Institute Methodes et composes pour accroitre la puissance des agents antifongiques
JP2019502753A (ja) 2015-12-23 2019-01-31 ムーンショット ファーマ エルエルシー ナンセンス変異依存ｍＲＮＡ分解機構の抑制による免疫応答の誘導法
EP3641752A4 (fr) 2017-06-22 2021-03-17 Moonshot Pharma LLC Méthodes de traitement du cancer au moyen de compositions comprenant de l'amlexanox et des immunomodulateurs
WO2019033249A1 (fr) * 2017-08-14 2019-02-21 深圳市博奥康生物科技有限公司 Sharn du gène btla humain et utilisation correspondante
CN109721657B (zh) * 2017-10-27 2021-11-02 北京比洋生物技术有限公司 阻断pd-1/pd-l1信号传导途径且活化t细胞的融合蛋白及其用途
US20190284553A1 (en) 2018-03-15 2019-09-19 KSQ Therapeutics, Inc. Gene-regulating compositions and methods for improved immunotherapy
JP2021536239A (ja) * 2018-08-28 2021-12-27 ロシュ イノベーション センター コペンハーゲン エーエス スプライス調節化合物を使用したネオアンチゲン操作
CN109971762B (zh) * 2019-04-24 2022-12-20 中国农业科学院上海兽医研究所(中国动物卫生与流行病学中心上海分中心) 日本血吸虫eIF4A基因的siRNA及其应用
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